Method for inducing immunity with a peptide fragment from human CAPRIN-1

ABSTRACT

The invention relates to an immunity-inducing agent comprising, as an active ingredient, at least one polypeptide having immunity-inducing activity that is selected from among polypeptides (a), (b), and (c): (a) a polypeptide of at least seven contiguous amino acids of the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing; (b) a polypeptide of at least seven amino acids having 90% or more sequence identity with the polypeptide (a); and (c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence thereof, or a recombinant vector comprising a polynucleotide encoding said polypeptide and capable of expressing said polypeptide in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 37 C.F.R. §1.53(b) divisional of U.S. application Ser. No. 13/056,122 filed on Jan. 27, 2011, which is a National Phase of PCT International Application No. PCT/JP2009/063881, filed on Aug. 5, 2009, and claims priority under 35 U.S.C. §119(a) to Japanese Application No. 2008-202065, filed in Japan on Aug. 5, 2008. The entire contents of all of the above applications are hereby expressly incorporated by reference.

TECHNICAL FIELD

The present invention relates to a novel immunity-inducing agent that is useful as a therapeutic and/or preventive agent for cancer or the like.

BACKGROUND ART

Cancer is the overall leading cause of death. At present, the primary form of cancer treatment technique is surgical treatment, which is carried out in combination with radiation treatment and chemotherapy. In spite of the development of novel surgical techniques and the discovery of novel anticancer agents of recent years, outcomes from cancer treatment still remain unimproved, except in the cases of some types of cancer. In recent years, cancer antigens recognized by cytotoxic T cells that are reactive to cancer and genes encoding cancer antigens have been identified along with the development of molecular biology and cancer immunology, and expectations for antigen-specific immunotherapy have increased (Tsuyoshi Akiyoshi, Gan to Kagaku Ryouhou (Cancer and Chemotherapy), 1997, vol. 24, pp. 551-519, Cancer and Chemotherapy Publishers Inc., Japan).

Immunotherapy requires the cancer-cell-specific presence of a peptide, polypeptide, or protein that is recognized as a target antigen, as well as substantial absence thereof in normal cells from the viewpoint of alleviation of side effects. In 1991, Boon et al. (the Ludwig Institute for Cancer Research, Belgium) isolated the human melanoma antigen MAGE1 recognized by the CD8+ T cell via cDNA expression cloning using an autologous cancer cell line and cancer-reactive T cells (Bruggen P. et al., Science, 254: 1643-1647, 1991). Thereafter, the SEREX (serological identifications of antigens by recombinant expression cloning) method, which identifies the tumor antigen recognized by the antibody produced in response to autologous cancer in the body of a cancer patient via gene expression cloning was reported (Proc. Natl. Acad. Sci. U.S.A., 92: 11810-11813, 1995; and U.S. Pat. No. 5,698,396). Some cancer antigens have been isolated by such techniques (Int. J. Cancer, 72: 965-971, 1997; Cancer Res., 58: 1034-1041, 1998; Int. J. Cancer, 29: 652-658, 1998; Int. J. Oncol., 14: 703-708, 1999; Cancer Res., 56: 4766-4772, 1996; and Hum. Mol. Genet. 6: 33-39, 1997). In addition, clinical testing of cancer immunotherapy targeting some such antigitens has been initiated.

As in the case of humans, dogs and cats are known to suffer from a variety of tumors, such as mammary gland cancer, leukemia, and lymphoma, and tumors are highly ranked in statistics for canine or feline diseases. However, there are no effective therapeutic, preventive, or diagnostic agents for canine or feline cancer at present. Most dog or cat owners would not notice canine or feline tumors until tumors become advanced and enlarged. Even if tumors are removed via surgical operation or drugs for human use (e.g., anticancer drugs) are administered, tumors are often already beyond cure, and animals often die shortly after treatment. Under such circumstances, if therapeutic, preventive, and diagnostic agents for cancer that are effective for dogs or cats become available, application thereof for canine or feline cancer can be expected.

The cytoplasmic and proliferation-associated protein 1 (CAPRIN-1) is expressed when dormant normal cells are activated or undergo cell division. CAPRIN-1 is an intracellular protein that is known to form intracellular stress granules with RNA in the cell and to be associated with regulation of mRNA transportation and translation. CAPRIN-1 is also known by various other names, and examples thereof include the GPI-anchored membrane protein 1 and the membrane component surface marker 1 protein (M11S1). CAPRIN-1 has names that convey the impression that it has been known as a cell membrane protein. Such other names derive from a report to the effect that the CAPRIN-1 gene sequence has a GPI-binding region and it is a membrane protein expressed in a large-intestine-derived cell line (J. Biol. Chem., 270: 20717-20723, 1995). Later, however, it was known that the CAPRIN-1 gene sequence in this report was incorrect, and in the gene sequence, deletion of a single nucleotide from the CAPRIN-1 gene sequence currently registered in the GenBank or the like causes a frame shift, thereby leading to deletion of 80 amino acids from the C terminus, and therefore the resulting artifact (74 amino acids) was the GPI-binding region mentioned in the foregoing report. In addition, it was also known that the CAPRIN-1 gene sequence shown in the report also had an error at the 5′ side, and 53 amino acid residues had been deleted from the N-terminus (J. Immunol., 172: 2389-2400, 2004). It has also been reported that the protein encoded by the CAPRIN-1 gene sequence currently registered in GenBank or the like is not a cell membrane protein (J. Immunol., 172: 2389-2400, 2004).

Based on the report of J. Biol. Chem., 270: 20717-20723, 1995, that CAPRIN-1 is a cell membrane protein, US 2008/0075722 and WO 2005/100998 describe that CAPRIN-1 can be a target of cancer therapy as a cell membrane protein under the name of M11S1. However, they do not include any specific descriptions in the Examples. As reported in J. Immunol., 172: 2389-2400, 2004, however, it has been heretofore accepted since US 2008/0075722 was filed that CAPRIN-1 is not expressed on a cell surface. It is apparent that the disclosures of US 2008/0075722 and WO 2005/100998 based only on the incorrect information that CAPRIN-1 is a cell membrane protein should not be understood as general technical knowledge in the art. In addition, there is no report that the expression level of CAPRIN-1 is higher in cancer cells such as breast cancer cells than in normal cells.

SUMMARY OF THE INVENTION Problem to Be Solved by the Invention

It is an object of the present invention to discover a novel polypeptide useful for a therapeutic and/or preventive agent for cancer and to provide use of such polypeptide as an immunity-inducing agent.

Means for Solving the Problem

The present inventors have conducted concentrated studies, and then they obtained cDNAs encoding proteins that bind to antibodies in the serum obtained from a cancer-bearing living body by the SEREX method using a canine-testicular-tissue-derived cDNA library and the serum of a dog afflicted with breast cancer, and they prepared a canine CAPRIN-1 polypeptide having the amino acid sequences shown by SEQ ID NOs: 6, 8, 10, 12, and 14 using such cDNAs. Using the human homologous gene of the obtained gene, also, they prepared a human CAPRIN-1 polypeptide having the amino acid sequences as shown by SEQ ID NOs: 2 and 4. Further, they found that such CAPRIN-1 polypeptides were expressed specifically in a breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophagus cancer, and colorectal cancer. Furthermore, they found that administration of such CAPRIN-1 polypeptides to living bodies would lead to induction of immunocytes against CAPRIN-1 polypeptides in the living bodies and regression of tumors in living bodies expressing the CAPRIN-1 genes. In addition, they found that the antibodies against such CAPRIN-1 polypeptides would disrupt cancer cells that express the CAPRIN-1 genes and induce antitumor effects in vivo. This has led to the completion of the present invention.

Accordingly, the present invention has the following features.

(1) An immunity-inducing agent comprising, as an active ingredient, at least one polypeptide having immunity-inducing activity and selected from the following polypeptides (a), (b), and (c), or a recombinant vector comprising a polynucleotide encoding such polypeptide and capable of expressing such polypeptide in vivo:

(a) a polypeptide of at least seven contiguous amino acids of the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing;

(b) a polypeptide of at least seven amino acids having 90% or more sequence identity with the polypeptide (a); and

(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence thereof.

(2) The immunity-inducing agent according to (1), wherein the polypeptide (b) is a polypeptide having 95% or more sequence identity with the polypeptide (a).

(3) The immunity-inducing agent according to (1), wherein the polypeptide having immunity-inducing activity is a polypeptide of at least seven contiguous amino acids of the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing or a polypeptide comprising such polypeptide as a partial sequence thereof.

(4) The immunity-inducing agent according to (3), wherein the polypeptide having immunity-inducing activity is a polypeptide comprising the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing.

(5) The immunity-inducing agent according to (3), wherein the polypeptide having immunity-inducing activity is a polypeptide of at least seven contiguous amino acids in the region of amino acid residues (aa) 41 to 400 or amino acid residues (aa) 503 to 564 of the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing except for SEQ ID NO: 6 and SEQ ID NO: 18 or a polypeptide comprising such polypeptide as a partial sequence thereof.

(6) The immunity-inducing agent according to (5), wherein the polypeptide having immunity-inducing activity is a polypeptide of the amino acid sequence shown by any of SEQ ID NOs: 43 to 76 in the Sequence Listing or a polypeptide of 8 to 12 amino acids comprising the amino acid sequence shown by any of SEQ ID NOs: 43 to 76 in the Sequence Listing as a partial sequence thereof.

(7) The immunity-inducing agent according to any of (1) to (6), which comprises, as an active ingredient, one or plural types of such polypeptides.

(8) The immunity-inducing agent according to (7), wherein the polypeptide is an agent for treating an antigen-presenting cell.

(9) The immunity-inducing agent according to any of (1) to (7), which is for use in the treatment or prevention of animal cancer.

(10) The immunity-inducing agent according to (9), wherein the cancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer, esophagus cancer, or colorectal cancer.

(11) The immunity-inducing agent according to (9), wherein the animal is a human, dog, or cat.

(12) The immunity-inducing agent according to any of (1) to (11), which further comprises an immunopotentiating agent.

(13) The immunity-inducing agent according to (12), wherein the immunopotentiating agent is at least one adjuvant or cytokine selected from the group consisting of Freund's incomplete adjuvant, Montanide, poly IC and a derivative thereof, CpG oligonucleotide, interleukin 12, interleukin 18, interferon α, interferon β, interferon ω, interferon γ, and Flt3 ligand.

(14) An isolated antigen-presenting cell comprising a complex of the above-mentioned polypeptide having immunity-inducing activity and an HLA molecule.

(15) An isolated T cell, which selectively binds to a complex of the above-mentioned polypeptide having immunity-inducing activity and an HLA molecule.

(16) A method for inducing immunity comprising administering to an individual at least one polypeptide having immunity-inducing activity and selected from the following polypeptides (a) to (c), or a recombinant vector comprising a polynucleotide encoding such polypeptide and capable of expressing such polypeptide in vivo:

(a) a polypeptide of at least seven contiguous amino acids of the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing;

(b) a polypeptide of at least seven amino acids having 90% or more sequence identity with the polypeptide (a); and

(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence thereof.

Effects of the Invention

The present invention provides a novel immunity-inducing agent useful for treatment and/or prevention of cancer. As specifically described in the examples below, administration of the polypeptide used in the present invention to a cancer-bearing animal enables induction of an immunocyte in the body of such cancer-bearing animal, which further enables shrinkage or regression of existing cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression pattern of the gene encoding the CAPRIN-1 polypeptide in normal tissue and a tumor cell line. Reference number 1 represents the expression pattern of the gene encoding the CAPRIN-1 protein, and Reference number 2 represents the expression pattern of the GAPDH gene.

In FIG. 2, Reference numbers 3 to 31 on the horizontal axis each represent the capacity of HLA-A0201+ CD8+ T cells for producing IFN-γ stimulated by the T2 cells pulsed with the peptides of SEQ ID NOs: 43 to 71. Reference number 32 represents the results regarding a negative control peptide of SEQ ID NO: 77 (a peptide having a sequence outside the scope of the present invention).

In FIG. 3, Reference numbers 33 to 37 on the horizontal axis each represent the capacity of the HLA-A24+ CD8+ T cells for producing IFN-γ stimulated by the JTK-LCL cells pulsed with the peptides of SEQ ID NOs: 72 to 76. Reference number 38 represents the results regarding a negative control of SEQ ID NO: 77.

In FIG. 4, Reference numbers 39 to 67 on the horizontal axis each represent the cytotoxic activity of the HLA-A0201+ CD8+ T cells stimulated with the use of the peptides of SEQ ID NOs: 43 to 71 on the U-87MG cells. Reference number 68 represents the cytotoxic activity of the CD8+ T cells induced with the use of a negative control peptide (SEQ ID NO: 77).

In FIG. 5, Reference numbers 69 to 73 on the horizontal axis each represent the cytotoxic activity of the HLA-A24+ CD8+ T cells stimulated with the use of the peptides of SEQ ID NOs: 72 to 76 on the JTK-LCL cells. Reference number 74 represents the cytotoxic activity of the CD8+ T cells induced with the use of a negative control peptide (SEQ ID NO: 77).

EMBODIMENTS FOR CARRYING OUT THE INVENTION Polypeptides

The polypeptides contained in the immunity-inducing agent of the present invention as an active ingredient include one or a plurality of polypeptides selected from the following polypeptides (a), (b), and (c):

(a) a polypeptide of at least seven contiguous amino acids in a polypeptide having the amino acid sequence shown by any even SEQ ID number selected from SEQ ID NOs: 2 to 30 listed in the Sequence Listing and having immunity-inducing activity;

(b) a polypeptide having 90% or more sequence identity with the polypeptide (a), consisting of at least 7 amino acids, and having immunity-inducing activity; and

(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence and having immunity-inducing activity.

The term “polypeptide” used herein refers to a molecule formed via peptide bonds among a plurality of amino acids. The term refers not only to a polypeptide molecule constituted by a large number of amino acids but also a low-molecular-weight molecule constituted by a small number of amino acids (an oligopeptide) and a full-length protein. In the present invention, the term “polypeptide” also refers to a protein of a full length sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30.

The nucleotide sequences of polynucleotides encoding separate proteins consisting of the amino acid sequences as shown by even SEQ ID numbers among SEQ ID NOs: 2 to 30 (i.e., SEQ ID NOs: 2, 4, 6 . . . 28, and 30) are shown by odd SEQ ID numbers among SEQ ID NOs: 1 to 29 (i.e., SEQ ID NOs: 1, 3, 5 . . . 27, and 29).

The term “having the amino acid sequence” used herein refers to a sequence composed of amino acid residues in a particular order. For example, the term “a polypeptide having the amino acid sequence shown by SEQ ID NO: 2” refers to a polypeptide of 709 amino acid residues in length possessing the amino acid sequence shown by SEQ ID NO: 2, i.e., Met Pro Ser Ala Thr . . . (snip) . . . Gln Gln Val Asn. The term “a polypeptide having the amino acid sequence shown by SEQ ID NO: 2” may be occasionally abbreviated as “the polypeptide of SEQ ID NO: 2.” The same applies to the expression “having the nucleotide sequence.” In the context of that, the term “having” is interchangeable with the expression “consisting of.”

The term “immunity-inducing activity” used herein refers to the capacity for inducing an immunocyte that secretes cytokine, such as interferon or interleukin, in vivo.

Whether or not a polypeptide has immunity-inducing activity can be confirmed via, for example, known ELISPOT assay. Specifically, cells such as peripheral blood mononuclear cells are obtained from a living body to which a polypeptide to be assayed for the immunity-inducing activity has been administered, such cells are co-cultured in the presence of such polypeptide, and the production amount of cytokine and/or chemokine, such as IFN-γ or interleukin (IL), from the cells is measured with the use of a specific antibody, as described in the Examples below, for example. Thus, the number of immunocytes among the cells can be assayed. This enables evaluation of immunity-inducing activity.

Alternatively, a recombinant polypeptide prepared based on an amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 may be administered to a cancer-bearing animal, so that a tumor can be regressed by the immunity-inducing activity, as described in the Examples below. Thus, the immunity-inducing activity can be evaluated as the capacity for suppressing the growth of cancer cells expressing a polypeptide shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 or the capacity for shrinking or eliminating cancer tissue (tumor) (hereafter, such capacity is referred to as “antitumor activity”). The antitumor activity of the polypeptides can be determined by, for example, actually administering such polypeptide to a cancer-bearing living body and examining whether or not the tumor is shrinked, as specifically described in the Examples below.

Alternatively, whether or not T cells stimulated by the polypeptide (i.e., T cells brought into contact with the antigen-presenting cells that present such polypeptide) exhibit cytotoxic activity on tumor cells in vitro may be examined to evaluate the antitumor activity of the polypeptide. T cells can be brought into contact with antigen-presenting cells via co-culture thereof in a liquid medium as described below. The cytotoxic activity can be assayed via a known technique referred to as the ⁵¹Cr-release assay technique described in, for example, Int. J. Cancer, 58: p. 317, 1994. When the polypeptides are used for treatment and/or prevention of cancer, it is preferable that the immunity-inducing activity be evaluated using the antitumor activity as an indicator, although a method of evaluation is not particularly limited.

The amino acid sequences shown by even SEQ ID numbers among SEQ ID NOs: 2 to 30 listed in the Sequence Listing disclosed by the present invention are the amino acid sequences of the CAPRIN-1 polypeptides isolated as the polypeptides binding to the antibodies existing specifically in the serum obtained from cancer-bearing dog and human, bovine, horse, mouse, and chicken homologues of such polypeptides by the SEREX method using the normal canine testicular tissue-derived cDNA library and the serum of a dog afflicted with breast cancer (see Example 1 below).

The polypeptide (a) indicated above is of at least 7 and preferably at least 8, 9, 10 or more contiguous amino acids in a polypeptide having an amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 and has immunity-inducing activity. Particularly preferably, such polypeptide has an amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30. As known in the art, a polypeptide of at least about 7 amino acid residues can exert antigenicity. Accordingly, a polypeptide of at least seven contiguous amino acid residues of the amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 can exert antigenicity and immunogenicity. That is, a polypeptide of at least seven contiguous amino acid residues of the amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 can have immunity-inducing activity, and such polypeptide can be used for preparing the immunity-inducing agent of the present invention. Based on the fact that antibodies produced against an antigenic substance in vivo are polyclonal antibodies, a polypeptide composed of a larger number of amino acid residues can induce a larger variety of antibodies recognizing various sites of the antigenic substance, thereby enhancing the immunity-inducing activity. In order to enhance immunity-inducing activity, accordingly, the number of amino acid residues may be preferably at least 30 or more, or 50 or more, more preferably at least 100 or more, 150 or more, and further preferably at least 200 or more, or still preferably 250 or more.

As the principle of immunity induction via administration of a cancer antigen polypeptide, it is known that a polypeptide is incorporated into an antigen-presenting cell, the polypeptide is degraded by a peptidase in the cell into a smaller fragment (hereafter it may be referred to as an “epitope”), such fragment is presented on the cell surface, cytotoxic T cells or the like recognize such fragment and selectively kill the antigen-presenting cells. The size of a polypeptide presented on the antigen-presenting cell surface is relatively small, and it is about 7 to 30 in terms of the number of amino acids. From the viewpoint of presentation on the antigen-presenting cell, accordingly, it is sufficient that the polypeptide (a) is of about 7 to 30 and preferably about 8 to 30 or 9 to 30 contiguous amino acids in the amino acid sequences shown by any even SEQ ID number among SEQ ID NOs: 2 to 30. Such polypeptide of a relatively small size may be directly presented on the antigen-presenting cell surface without being incorporated into the antigen-presenting cell.

The polypeptide incorporated into the antigen-presenting cell is cleaved at random positions with a peptidase present in the cells, a variety of polypeptide fragments are generated, and such polypeptide fragments are presented on the antigen-presenting cell surface. If a large polypeptide such as a full-length sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 is administered, accordingly, polypeptide fragments that are effective for immunity induction mediated by antigen-presenting cells via degradation in the antigen-presenting cell are naturally generated. Thus, a large-size polypeptide can be preferably used for immunity induction mediated by antigen-presenting cells, and the number of amino acids may be at least 30, more preferably at least 100, further preferably at least 200, and still further preferably at least 250.

Further, the polypeptide of the present invention can be screened for a peptide being a possible epitope with the use of a matching medium that can search for a peptide serving as a possible epitope having a binding motif for each HLA type, such as the HLA Peptide Binding Predictions of Bioinformatics & Molecular Analysis Selection (BIMAS) (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html). Specifically, a polypeptide of at least seven contiguous amino acids in the region of amino acid residues (aa) 41 to 400 or amino acid residues (aa) 503 to 564 in the amino acid sequences shown by any even SEQ ID number selected from among SEQ ID NOs: 2 to 30 except for SEQ ID NO: 6 and SEQ ID NO: 18 or a polypeptide comprising such polypeptide as a partial sequence thereof is preferable. In the polypeptide of SEQ ID NO: 2, a polypeptide shown by any of SEQ ID NOs: 43 to 76 is more preferable.

The polypeptide (b) above is derived from the polypeptide (a) by substitution, deletion, addition, and/or insertion of a small number of (preferably one or several) amino acid residues, it has 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, further preferably 98% or more, 99% or more, or 99.5% or more sequence identity with the original sequence, and it has immunity-inducing activity. When a small number of (preferably one or several) amino acid residues are substituted with, deleted from, added to, or inserted into the amino acid sequence of the protein antigen, in general, it is extensively known in the art that the resulting protein occasionally has substantially the same antigenicity or immunogenicity with that of the original protein. Thus, the polypeptide (b) above is capable of exerting the immunity-inducing activity and it can be thus used for preparing the immunity-inducing agent of the present invention. Alternatively, the polypeptide (b) above is preferably a polypeptide having an amino acid sequence derived from the amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 by substitution, deletion, addition, and/or insertion of one or several amino acid residues. The term “several” used herein refers to an integer from 2 to 10, preferably an integer from 2 to 6, and further preferably an integer from 2 to 4.

The term “sequence identity” used herein regarding the amino acid sequence or nucleotide sequence represents a percentage (%) determined by aligning two amino acid sequences (or nucleotide sequences) to be compared so as to maximize the number of matching amino acid residues (or nucleotides) and dividing the number of matched amino acid residues (or the number of matched nucleotides) by the total number of amino acid residues (or the total number of nucleotides). When aligning the sequences as described above, a gap is adequately inserted into one or both of the two sequences to be compared, according to need. Such sequence alignment can be carried out with the use of a well-known program, for example, BLAST, FASTA, or CLUSTAL W (Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., 87: 2264-2268, 1993; Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997). When a gap is inserted, the total number of amino acid residues (or the total number of nucleotides) is the number of residues (or the number of nucleotides) counted by designating a gap as an amino acid residue (or a nucleotide). When the total number of amino acid residues (or the total number of nucleotides) thus determined differs between the two sequences to be compared, identity (%) is determined by dividing the number of the matched amino acid residues (or the number of nucleotides) by the total number of amino acid residues (or the total number of nucleotides) of a longer sequence.

A preferable amino acid substitution is a conservative amino acid substitution. Twenty types of amino acids constituting a naturally-occurring protein can be classified into groups of amino acids having similar properties: i.e., neutral amino acids having low-polarity side chains (Gly, Ile, Val, Leu, Ala, Met, and Pro); neutral amino acids having hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, and Cys); acidic amino acids (Asp, and Glu); basic amino acids (Arg, Lys, and His); and aromatic amino acids (Phe, Tyr, Trp, and His). It is known that substitution within such groups; i.e., conservative substitution, would not alter polypeptide properties in many cases. When amino acid residues in the polypeptide (a) of the present invention are substituted, accordingly, substitution may be carried out within such groups, so that a possibility of maintaining the immunity-inducing activity can be enhanced. In the present invention, however, the altered polypeptide may have non-conservative substitution, provided that the resulting polypeptide has immunity-inducing activity equivalent or substantially equivalent to that of an unaltered polypeptide.

The polypeptide (c) comprises the polypeptide (a) or (b) as a partial sequence thereof and has immunity-inducing activity. Specifically, the polypeptide (c) corresponds to the polypeptide (a) or (b) to which other amino acid(s) or polypeptide(s) are added at one or both ends thereof and having immunity-inducing activity. Such polypeptide can be used for preparing the immunity-inducing agent of the present invention.

The above-mentioned polypeptide can be chemically synthesized in accordance with, for example, the Fmoc (fluorenylmethyloxycarbonyl) method or the tBoc (t-butyloxycarbonyl) method (the Japanese Biochemical Society (ed.), Seikagaku Jikken Kouza (the Course for Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of Protein) IV, Kagaku Shushoku to Peptide Gousei (Chemical Modification and Peptide Synthesis), Tokyo Kagaku Dojin, Japan, 1981). Also, a variety of commercially available peptide synthesizers can be used to synthesize the polypeptide in accordance with a conventional technique. Further, known genetic engineering techniques (e.g., Sambrook et al., Molecular Cloning, vol. 2, Current Protocols in Molecular Biology, 1989, Cold Spring Harbor Laboratory Press; and Ausubel et al., Short Protocols in Molecular Biology, vol. 3, A compendium of Methods from Current Protocols in Molecular Biology, 1995, John Wiley & Sons) may be employed to prepare a polynucleotide encoding the above polypeptide, the resulting polypeptide may be incorporated into an expression vector and then introduced into a host cell, and the polypeptide may be produced in such host cell to obtain the target polypeptide.

A polynucleotide encoding the above polypeptide can be easily prepared via a known genetic engineering technique or a conventional technique using a commercially available nucleic acid synthesizer. For example, DNA having the nucleotide sequence of SEQ ID NO: 1 can be prepared by performing PCR with the use of the human chromosome DNA or cDNA library as a template and a pair of primers designed so as to amplify the nucleotide sequence shown by SEQ ID NO: 1. Similarly, DNA having the nucleotide sequence of SEQ ID NO: 5 can be prepared with the use of the canine chromosome DNA or cDNA library as the template. PCR conditions can be adequately determined. For example, a reaction cycle of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds to 1 minute, and extension at 72° C. for 2 minutes with the use of thermostable DNA polymerase (e.g., Taq polymerase) and Mg²⁺-containing PCR buffer is repeated 30 times, followed by the reaction at 72° C. for 7 minutes, although the reaction conditions are not limited thereto. PCR techniques, conditions, and the like are described in, for example, Ausubel et al., Short Protocols in Molecular Biology, vol. 3, A compendium of Methods from Current Protocols in Molecular Biology, 1995, John Wiley & Sons (Chapter 15, in particular). Also, adequate probes or primers may be prepared based on the information of the nucleotide sequences and the amino acid sequences shown by SEQ ID NOs: 1 to 30 in the Sequence Listing of the present invention, and human, canine, bovine, or other cDNA libraries may be screened for with the use of such probes or primers, so that DNA of interest can be isolated. cDNA libraries are preferably prepared from cells, organs, or tissue in which a protein shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 is expressed. Procedures, such as preparation of probes or primers, construction of cDNA library, screening of cDNA library, and cloning of target genes, described above are known in the art. For example, such procedures can be carried out in accordance with the methods described in Sambrook et al., Molecular Cloning, vol. 2, Current Protocols in Molecular Biology, 1989), Ausubel et al. (as above). DNA encoding polypeptide (a) above can be obtained from DNA thus obtained. Since a codon encoding each amino acid is known, a nucleotide sequence of a polynucleotide encoding a particular amino acid sequence can be easily identified. Accordingly, the nucleotide sequence of a polynucleotide encoding polypeptide (b) or (c) can be easily identified, and such polynucleotide can also be easily synthesized with the use of a commercially available nucleic acid synthesizer in accordance with a conventional technique.

The host cells may be any cells, provided that the aforementioned polypeptide can be expressed therein. The host cells include, but not limited to, an E. coli cell as prokaryotic cells; and monkey kidney cells (COS 1), chinese hamster ovary (CHO) cells, the human embryonic kidney cell line (HEK293), and the fetal mouse skin cell line (NIH3T3), budding yeast cells, dividing yeast cells, silk worm cells, and xenopus egg cells as eukaryotic cells

When prokaryotic host cells are used, expression vectors having, for example, an origin, a promoter, a ribosome-binding site, a multi-cloning site, a terminator, a drug-tolerant gene, and an auxotrophic complementary gene that can be replicated in prokaryotic cells, are used. Examples of E. coli expression vectors include pUC, pBluescriptII, the pET expression system, and the pGEX expression system. DNA encoding the above polypeptide may be incorporated into such expression vector, prokaryotic host cells may be transformed with such vector, and the resulting transformant may be cultured. Thus, a polypeptide encoded by the DNA can be expressed in prokaryotic host cells. In this case, such polypeptide can be expressed in the form of a fusion protein with another protein.

When eukaryotic host cells are used, eukaryotic cell expression vectors having, for example, a promoter, a splicing region, and a poly(A) addition site are used. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV, pRS, pcDNA3, and pYES2 vectors. As described above, DNA encoding the above polypeptide may be incorporated into such expression vector, eukaryotic host cells may be transformed with such vector, and the resulting transformant may then be cultured. Thus, a polypeptide encoded by the DNA can be expressed in eukaryotic host cells. When pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, or other expression vectors are used, the polypeptide can be expressed in the form of a fusion protein with a variety of tags, such as His tag (e.g., (His)₆ to (His)₁₀), FLAG tag, myc tag, HA tag, or GFP.

Expression vectors can be introduced into host cells via conventional techniques, such as electroporation, the calcium phosphate method, the liposome method, the DEAE-dextran method, microinjection, virus infection, lipofection, or binding with a cell-permeable peptide.

The target polypeptide can be isolated and purified from host cells by employing known separation techniques in combination. Examples thereof include, but are not limited to, treatment with the use of a denaturing agent such as urea or a surfactant, ultrasonication, enzyme digestion, salting out or fractional precipitation with a solvent, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.

Some polypeptides obtained by such methods are in the form of fusion proteins with any other proteins as described above. Examples thereof include fusioin proteins with glutathione-S-transferase (GST) or His tag. Such polypeptides in the form of fusion proteins are within the scope of the present invention as polypeptide (c). Further, polypeptides expressed in transformed cells are translated, and the translated polypeptides occasionally undergo various types of modification in the cells. Such post-translationally modified polypeptides are also within the scope of the present invention, provided that such polypeptides have immunity-inducing activity. Examples of such post-translational modification include elimination of N-terminal methionine, N-terminal acetylation, sugar chain addition, limited degradation with intracellular protease, myristoylation, isoprenylation and phosphorylation.

<Immunity-Inducing Agent>

As specifically described in the examples below, administration of the above-mentioned polypeptide having immunity-inducing activity to a cancer-bearing living body enables regression of an existing tumor. Thus, the immunity-inducing agent of the present invention can be used as a therapeutic and/or preventive agent for cancer.

The terms “tumor” and “cancer” used herein refer to malignant neoplasms, and these terms are used interchangeably with each other.

In this case, target cancers are those expressing a gene encoding a polypeptide comprising an amino acid sequence shown by any even SEQ ID number among SEQ ID NOs: 2 to 30 or a partial sequence thereof consisting of at least 7 contiguous amino acids. Preferably, such cancers are breast cancer, brain tumor, leukemia, lung cancer, lymphoma, mast cell tumor, esophagus cancer, and colorectal cancer. Examples of such specified cancers include, but are not limited to, mammary gland cancer, combined mammary gland cancer, malignant mixed tumor of the mammary gland, intraductal papillary adenocarcinoma, chronic lymphocytic leukemia, gastrointestinal lymphoma, digestive lymphoma, and small to medium cell lymphoma.

Target animals are mammalians, and examples thereof include mammalian animals, including primates, pet animals, livestock animals, and competitive animals, with humans, dogs, and cats being particularly preferable.

The immunity-inducing agent of the present invention may be administered orally or parenterally to an organism. Parenteral administration, such as intramuscular, subcutaneous, intravenous, or intraarterial administration, is preferable. When such immunity-inducing agent is used for the purpose of cancer treatment, the agent can be administered to the regional lymph node in the vicinity of the tumor to be treated, so as to improve the antitumor effects as described in the examples below. The dose may be any amount as long as it is effective for immunity induction. When the agent is used for treatment and/or prevention of cancer, for example, an amount effective for treatment and/or prevention of cancer is sufficient, and such amount can be altered depending on, for example, the body weight, sex (i.e., male or female), or symptom of an animal. The amount effective for treatment and/or prevention of cancer is adequately determined in accordance with the tumor size, symptoms, or other conditions. In general, an effective amount for a target animal per day is 0.0001 μg to 1,000 μg, and preferably 0.001 μg to 1,000 μg, and the agent can be administered via a single dose or a plurality of doses. Preferably, the agent is administered via several separate doses every several days or months. As specifically described in the examples below, the immunity-inducing agent of the present invention enables regression of an existing tumor. Thus, the agent can exert the antitumor effects on a small number of cancer cells in an early developmental stage. Use thereof before the onset of cancer or after treatment leads to prevention of the development or recurrence of cancer. Specifically the immunity-inducing agent of the present invention is useful for treatment and prevention of cancer.

The immunity-inducing agent of the present invention may consist of a polypeptide, or it may be adequately mixed with an additive that is suitable for a relevant dosage form, such as a pharmaceutically acceptable carrier, a diluent, an excipient, or the like. Methods of preparing an agent and additives that can be used are well-known in the medical preparation field, and any methods and additives can be employed. Specific examples of additives include, but are not limited to: diluents, such as physiological buffer solutions; excipients, such as sugar, lactose, corn starch, calcium phosphate, sorbitol, and glycine; binders, such as syrup, gelatin, gum Arabic, sorbitol, polyvinyl chloride, and tragacanth; and lubricants, such as magnesium stearate, polyethylene glycol, talc, and silica. Examples of forms of preparations include oral agents, such as tablets, capsules, granules, powders, and syrup solutions, and parenteral agents, such as inhalants, injection preparations, suppositories, and liquid drugs. Such agents can be prepared by common methods.

The immunity-inducing agent of the present invention can be used in combination with an immunopotentiating agent capable of potentiating an immunological response in vivo.

The immunopotentiating agent may be incorporated into the immunity-inducing agent of the present invention, or it may be administered to a patient as another composition in combination with the immunity-inducing agent of the present invention.

The term “patient” used herein refers to an animal, a mammalian animal in particular, and it is preferably a human, dog, or cat.

An example of the immunopotentiating agent is an adjuvant. An adjuvant provides an antigen reservoir (outside the cell or in the macrophage), it activates the macrophage, and it stimulates lymphocytes in a given tissue. Thus, an adjuvant can potentiate an immunological response and enhance the antitumor effects. When the immunity-inducing agent of the present invention is used for treatment and/or prevention of cancer, accordingly, it is particularly preferable that the immunity-inducing agent further comprise an adjuvant in addition to the polypeptide as an active ingredient. Various types of adjuvants are well-known in the art, and any such adjuvants can be used. Specific examples thereof include: MPL (SmithKline Beecham); an equivalent obtained by purification and acid hydrolysis of a lipopolysaccharide of Salmonella minnesota Re 595; QS21 (SmithKline Beecham); a pure saponin QA-21 purified from the Quillja saponaria extract; DQS21 disclosed in the PCT application (WO 96/33739, SmithKline Beecham); QS-7, QS-17, QS-18, and QS-L1 (So et al., Molecules and Cells, 1997, 7: 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; vitamin E; Montanide; alum; CpG oligonucleotide (e.g., Kreig et al., Nature, 1995, 374: 546-549); poly IC and a derivative thereof (e.g., poly ICLC); and various water-in-oil emulsions prepared from biodegradable oil, such as squalene and/or tocopherol. Freund's incomplete adjuvant, Montanide, poly I:C, a derivative thereof, and CpG oligonucleotide are particularly preferable. The rate of the adjuvant mixed with a polypeptide is typically about 1:10 to 10:1, preferably about 1:5 to 5:1, and further preferably about 1:1. It should be noted that adjuvants are not limited to those exemplified above, and other adjuvants known in the art can also be used at the time of administration of the immunity-inducing agent of the present invention (e.g., Goding, Monoclonal Antibodies: Principles and Practice, vol. 2, 1986). A method for preparing a mixture of polypeptide and adjuvant or an emulsion is well-known to a person skilled in the field of immunization.

As the immunopotentiating agent, factors that stimulate an immunological response of interest can be used in addition to the aforementioned adjuvants. For example, various cytokines that stimulate lymphocytes or antigen-presenting cells can be used as the immunopotentiating agent in combination with the immunity-inducing agent of the present invention. Many cytokines that can potentiate immunological responses are known in the art. Examples thereof include, but are not limited to, interleukin-12 (IL-12), GM-CSF, IL-18, interferon α, interferon β, interferon ω, interferon γ, and Flt3 ligand that are known to potentiate the protective effects of a vaccine. Such factor can be used as the immunopotentiating agent and can be administered to a patient in the form of a mixture thereof with the immunity-inducing agent of the present invention or in combination with the immunity-inducing agent of the present invention as another composition.

<Antigen-Presenting Cell>

Further, the above-mentioned polypeptides may be brought into contact with antigen-presenting cells in vitro to present such polypeptides to the antigen-presenting cells. Specifically, the polypeptides (a) to (c) can be used as agents for treating the antigen-presenting cells. Examples of antigen-presenting cells include dendritic cells, B cells, and macrophages, and dendritic cells or B cells having MHC class I molecules are preferably used. A variety of MHC class I molecules have been identified and well-known. Human MHC molecules are referred to as “HLA.” Examples of HLA class I molecules include HLA-A, HLA-B, and HLA-C. Specific examples include HLA-A1, HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, and HLA-Cw0602.

Dendritic cells or B cells having MHC class I molecules can be prepared from the peripheral blood by a well-known technique. For example, dendritic cells are induced from the bone marrow, umbilical blood, or peripheral blood of a patient with the use of the granulocyte-macrophage colony-stimulating factors (GM-CSF) and IL-3 (or IL-4), and tumor-associated peptides are added to the culture system. Thus, tumor-specific dendritic cells can be induced.

Administration of an effective amount of such dendritic cells enables induction of a response desirable for cancer treatment. Examples of cells that can be used include the bone marrow and the umbilical blood provided by a healthy individual and the bone marrow and the peripheral blood of the patient. When the patient's own autologous cells are used, a safety level is high, and serious side effects can be avoided. The peripheral blood or bone marrow may be a fresh, hypothermically stored, or cryopreserved sample. The peripheral blood may be prepared by culturing the whole blood or by culturing the separated leukocyte components, with the latter being preferable from the viewpoint of efficiency. Further, mononuclear cells may be isolated from the leukocyte components. When the sample is prepared from the bone marrow or umbilical blood, the entire cells that constitute the bone marrow may be cultured, or mononuclear cells may be separated therefrom and cultured. The peripheral blood, the leukocyte component thereof, and the bone marrow cells comprise mononuclear cells, hematopoietic stem cells, immature dendritic cells, or CD4+ cells from which dendritic cells originate. Cytokines may be of a naturally-occurring or gene recombinant type, and methods for producing the same are not limited, provided that safety and physiological activities thereof have been verified. Preferably, the minimum requirement of samples with verified medical qualities is used. The concentration of cytokine added is not particularly limited, provided that dendritic cells are induced. In general, the total cytokine concentration of approximately 10 to 1,000 ng/ml is preferable, and about 20 to 500 ng/ml is further preferable. Culture can be conducted with the use of a well-known medium that is generally used for leukocyte culture. A culture temperature is not particularly limited, provided that leukocytes can be multiplied, and the human body temperature (i.e., approximately 37° C.) is the most preferable. A gaseous environment during culture is not particularly limited, provided that leukocytes can be multiplied. Aeration with 5% CO₂ is preferable. Further, a culture duration is not particularly limited, provided that a necessary number of cells is induced. It is generally 3 days to 2 weeks. An adequate apparatus can be used for cell separation or culture, and it is preferable that such apparatus have the approved medical safety and stable and simple operability. In particular, cell culture apparatuses are not limited to common containers, such as petri-dishes, flasks, and bottles, and laminated or multistage containers, roller bottles, spinner bottles, bag-type culture apparatus, hollow fiber columns, or the like can also be used.

The above-mentioned polypeptides can be brought into contact with antigen-presenting cells in vitro via a well-known technique. For example, antigen-presenting cells can be cultured in a coculture solution containing such polypeptides. Peptide concentration in a medium is not particularly limited. In general, it is about 1 to 100 μg/ml, and preferably about 5 to 20 μg/ml. Cell density during culture is not particularly limited, and it is generally about 10³ to 10⁷ cells/ml, and preferably about 5×10⁴ to 5×10⁶ cells/ml. It is preferable that culture be conducted at 37° C. in 5% CO₂ in accordance with a conventional technique. A peptide length that can be presented on the antigen-presenting cell surface is generally about 30 amino acid residues at maximum. When antigen-presenting cells are brought into contact with polypeptides in vitro, accordingly, the length of the polypeptide may be adjusted to about 30 amino acid residues or less, although the length is not particularly limited thereto.

By culturing antigen-presenting cells in the presence of the polypeptides, peptides are incorporated into MHC molecules of the antigen-presenting cells and presented on the surfaces thereof. Thus, the isolated antigen-presenting cells containing the complex of polypeptides and MHC molecules can be prepared with the use of such polypeptides. Such antigen-presenting cells can present the polypeptides to T cells in vivo or in vitro, induce cytotoxic T cells specific for the polypeptides, and multiply such T cells.

The thus-prepared antigen-presenting cells containing the complex of polypeptides and MHC molecules may be brought into contact with T cells in vitro, so that cytotoxic T cells specific for such polypeptides can be induced and multiplied. It can be achieved by culturing the antigen-presenting cells together with the T cells in a liquid medium. For example, culture can be conducted by suspending antigen-presenting cells in a liquid medium, introducing the resulting suspension into a container such as wells of a microplate, and adding T cells thereto. The mixing ratio of antigen-presenting cells to T cells at the time of coculture is not particularly limited, and it is generally about 1:1 to 1:10, and preferably about 1:5 to 1:20 in terms of the cell count. Also, the density of antigen-presenting cells suspended in a liquid medium is not particularly limited, and it is generally about 100 to 10⁷ cells/ml, and preferably about 10⁴ to 10⁶ cells/ml. Co-culture is preferably carried out in accordance with a conventional technique at 37° C. in 5% CO₂. A culture duration is not particularly limited, and it is generally 2 days to 3 weeks, and preferably about 4 days to 2 weeks. It is preferable that coculture be carried out in the presence of a single type or a plurality of types of interleukins, such as IL-2, IL-6, IL-7, and IL-12. In such a case, IL-2 or IL-7 concentration is generally about 5 U/ml to 20 U/ml, IL-6 concentration is generally about 500 U/ml to 2,000 U/ml, and IL-12 concentration is generally about 5 ng/ml to 20 ng/ml, although the concentration is not limited thereto. The unit “U” used herein indicates a unit of activity. Co-culture may be repeated once or several times with the addition of fresh antigen-presenting cells. For example, the culture supernatant after coculture is discarded, coculture is further carried out with the addition of a suspension of fresh antigen-presenting cells, and such procedure may be repeated once or several times. Coculture conditions may be as described above.

Cytotoxic T cells specific for the polypeptides are induced and multiplied via the co-culture. Thus, isolated T cells that selectively bind to the complex of polypeptides and MHC molecules can be prepared with the use of the above polypeptides.

As described in the Examples below, genes encoding polypeptides of any even SEQ ID number among SEQ ID NOs: 2 to 30 are expressed specifically in the breast cancer cells, the leukemia cells, and the lymphoma cells. Accordingly, it is considered that a significantly larger number of polypeptides of even SEQ ID numbers among SEQ ID NOs: 2 to 30 are present in such cancer cells than in normal cells. When some polypeptides in cancer cells are presented to the MHC molecules on the cancer cell surface and the cytotoxic T cells prepared as described above are administered into a living body, cytotoxic T cells can disrupt cancer cells using the same as a marker. Since the antigen-presenting cells presenting the polypeptides are capable of inducing and multiplying cytotoxic T cells specific for the polypeptides in vivo, administration of the antigen-presenting cells into a living body can also disrupt cancer cells. That is, the cytotoxic T cells prepared with the use of the polypeptides or the antigen-presenting cells are useful as the therapeutic and/or preventive agent for cancer as with the immunity-inducing agent of the present invention.

When the isolated antigen-presenting cells or isolated T cells are administered to a living body, it is preferable that such isolated cells are prepared from the antigen-presenting cells or T cells sampled from the patient who receives the treatment with the use of the polypeptides (a) to (c) in order to avoid an immunological response that recognize such cells as foreign matter and attacks such cells in vivo.

The route of administration of a therapeutic and/or preventive agent for cancer comprising, as an active ingredient, the antigen-presenting cells or isolated T cells is preferably a parenteral route, such as intravenous or intraarterial administration. A dosage is adequately selected in accordance with the symptom, the purpose of administration, and other conditions, and in general, 1 to 10¹³ cells, and preferably 10⁶ to 10⁹ cells are used for administration, and such cells are preferably administered once every several days or several months. A preparation may be, for example, a suspension of cells in a physiological buffered saline solution, and it can be used in combination with other antitumor agents or cytokines. Also, one or more additives well-known in the medical preparation field can be added.

<Gene-Based Vaccine>

Polynucleotides encoding the polypeptides (a) to (c) may be expressed in the body of a target animal, so that antibody production or cytotoxic T cells can be induced in the body, and effects equivalent to those attained via polypeptide administration can be attained. Specifically, the immunity-inducing agent of the present invention may comprise polynucleotides encoding the polypeptides (a) to (c) and comprise, as an active ingredient, a recombinant vector capable of expressing such polypeptide in vivo. Such recombinant vector capable of expressing an antigen polypeptide is also referred to as a “gene-based vaccine.”

A vector used for preparing a gene-based vaccine is not particularly limited, provided that it can express polypeptides of interest in the target animal cells (preferably mammalian animal cells). It may be a plasmid or virus vector, and any vector known in the gene-based vaccine field may be used. Polynucleotides, such as DNA or RNA encoding the polypeptides, can be easily prepared in accordance with a conventional technique as described above. Also, the polynucleotides can be incorporated into a vector by a method well-known in the art.

Preferably, a gene-based vaccine is administered parenterally (e.g., intramuscular, subcutaneous, intravenous, or intraarterial administration), and the dosage can be adequately selected in accordance with an antigen type or other conditions. A dosage is generally about 0.1 μg to 100 mg, and preferably about 1 μg to 10 mg, in terms of the weight of the gene-based vaccine per kg of the body weight.

Examples of methods involving the use of virus vectors include methods in which the polynucleotide encoding the above polypeptide is incorporated into the RNA virus or DNA virus, such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, or Sindbis virus, and the target animal is infected therewith. Methods involving the use of retrovirus, adenovirus, adeno-associated virus, or vaccinia virus are particularly preferable.

Examples of other methods include a method in which an expression plasmid is directly administered into the muscle (the DNA vaccine method), the liposome method, the Lipofectin method, microinjection, the calcium phosphate method, and electroporation, with the DNA vaccine method and the liposome method being particularly preferable.

The gene encoding the polypeptide used in the present invention is actually allowed to function as a pharmaceutical product by the in vivo method in which the gene is introduced directly into the body or the ex vivo method in which a given cell is sampled from a target animal, the gene is introduced into the cell ex vivo, and the cell is then returned into the body (Nikkei Science (the Japanese version of Scientific American), April 1994, pp. 20-45, Japan; Gekkan Yakuji (the Pharmaceuticals Monthly), 1994, vol. 36, No. 1, pp. 23-48, Japan; Jikken Igaku Zoukan (an extra number of Experimental Medicine), 1994, vol. 12, No. 15, Japan; and cited documents thereof). The in vivo method is more preferable.

When a pharmaceutical agent is administered via the in vivo method, the agent can be administered through an adequate route in accordance with diseases, symptoms, and other conditions of the target of treatment. For example, administration can be carried out intravenously, intraarterially, subcutaneously, or intramuscularly. When the agent is administered via the in vivo method, for example, the agent can be in the form of a liquid drug. In general, the agent is in the form of an injection preparation containing DNA encoding the peptide of the present invention as an active ingredient, and common carriers may be added according to need. Also, the liposome or membrane fusion liposome (e.g., hemagglutinating virus of Japan (HVJ)-liposome) comprising the DNA can be in the form of a liposome preparation such as a suspension, cryogen, or cryogen condensed by centrifugation.

In the present invention, the term “the nucleotide sequence shown by SEQ ID NO: 1” refers not only to the nucleotide sequence that is actually shown by SEQ ID NO: 1 but also a sequence complementary thereto. Accordingly, the term “a polynucleotide having the nucleotide sequence shown by SEQ ID NO: 1” refers to a single-stranded polynucleotide having the nucleotide sequence that is actually shown by SEQ ID NO: 1, a single-stranded polynucleotide comprising a nucleotide sequence complementary thereto, and a double-stranded polynucleotide comprised of such single-stranded polynucleotides. When preparing a polynucleotide encoding the polypeptide used in the present invention, an adequate nucleotide sequence is to be selected. A person skilled in the art would readily select such adequate sequence.

EXAMPLES

Hereafter, the present invention is described in greater detail with reference to the Examples, although the technical scope of the present invention is not limited to the concrete examples below.

Example 1 Acquisition of Novel Cancer Antigen Protein by the SEREX Method

(1) Preparation of cDNA Library

Total RNA was extracted from the testicular tissue of a healthy dog by the acid guanidium-phenol-chloroform method, and poly(A) RNA was purified with the use of the Oligotex-dT30 mRNA purification Kit (Takara Shuzo Co., Ltd.) in accordance with the protocols included in the kit.

The canine testis cDNA phage library was synthesized using the obtained mRNA (5 μg). The cDNA phage library was prepared using the cDNA Synthesis Kit, theZAP-cDNA Synthesis Kit, and the ZAP-cDNA GigapackIII Gold Cloning Kit (STRATAGENE) in accordance with the protocols included in the kits. The size of the prepared cDNA phage library was 7.73×10⁵ pfu/ml.

(2) Screening of cDNA Library with the Use of Serum

Immunoscreening was carried out using the canine testis cDNA phage library prepared above. Specifically, host E. coli cells (XL1-Blue MRF′) was infected with the phage library in 2,210 clones per a NZY agarose plate (Φ90×15 mm), cultured at 42° C. for 3 to 4 hours to form plaques, the plate was covered with the nitrocellulose membrane (Hybond C Extra: GE Healthcare Bio-Science) infiltrated with IPTG (isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours to induce and express proteins, and the proteins were transferred onto the membrane. Thereafter, the membrane was recovered, immersed in TBS containing 0.5% dried skim milk (10 mM Tris-HCl, 150 mM NaCl, pH 7.5), and shaken at 4° C. overnight to block a non-specific reaction. The filter was allowed to react with the 500-fold diluted serum of a clinically affected dog at room temperature for 2 to 3 hours.

As the serum of the clinically affected dog, the serum sampled from a dog with breast cancer was used. The serum samples were stored at −80° C. and pretreated immediately before use. The serum samples were pretreated in the following manner. Specifically, host E. coli cells (XL1-BLue MRF′) were infected with the λ ZAP Express phages into which no foreign genes had been introduced, and culture was then conducted on the NZY plate medium at 37° C. overnight. Subsequently, 0.2 M NaHCO₃ buffer (pH 8.3) containing 0.5M NaCl was added to the plate, the plate was allowed to stand at 4° C. for 15 hours, and the supernatant was recovered as an E. coli/phage extract. Subsequently, the recovered E. coli/phage extract was allowed to flow through the NHS-column (GE Healthcare Bio-Science), and proteins derived from E. coli/phages were immobilized thereon. The serum of a clinically affected dog was allowed to flow through the protein-immobilized column for reacting therewith, and antibodies that had adsorbed to E. coli and phages were removed from the serum. The serum fraction that had flowed through the column was diluted 500-fold with TBS containing 0.5% dried skim milk, and the resultant was used as an immunoscreening sample.

The membrane onto which such treated serum and above-mentioned fusion protein had been blotted was washed 4 times with TBS-T (0.05% Tween 20/TBS), the goat anti-dog IgG (goat anti-dog IgG-h+I HRP conjugated: BETHYL Laboratories), which had been diluted 5,000-fold with TBS containing 0.5% dried skim milk as the secondary antibody, was allowed to react therewith at room temperature for 1 hour, detection was carried out via the enzymatic color-developing reaction using the NBT/BCIP reaction solution (Roche), colonies that correspond to the region positive for the color-development were removed from the NZY agarose plate (Φ90×15 mm), and the removed colonies were dissolved in 500 μl of SM buffer (100 mM NaCl, 10 mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin, pH 7.5). The secondary and the tertiary screenings were repeated until the color-development-positive colonies represented single clones in the same manner as described above, and 5 positive clones were isolated as a result of screening 30,940 phage clones that react with IgG in the serum.

(3) Homology Search of Isolated Antigen Gene

In order to subject the 5 positive clones isolated in the above-described manner to nucleotide sequence analysis, phage vectors were converted into plasmid vectors. Specifically, 200 μl of a solution of host E. coli (XL1-Blue MRF′) adjusted at OD₆₀₀ of 1.0 was mixed with 250 μl of the purified phage solution and 1 μl of ExAssist helper phage (STRATAGENE), the resultant was subjected to the reaction at 37° C. for 15 minutes, 3 ml of LB medium was added, culture was conducted at 37° C. for 2.5 to 3 hours, the culture product was incubated in a water bath at 70° C. for 20 minutes immediately thereafter, the resultant was centrifuged at 4° C. and 1,000×g for 15 minutes, and the supernatant was recovered as a phagemid solution. Subsequently, 200 μl of a solution of phagemid host E. coli (SOLR) adjusted at OD₆₀₀ of 1.0 was mixed with 10 μl of the purified phage solution, the resultant was subjected to the reaction at 37° C. for 15 minutes, 50 μl thereof was seeded on LB agar medium containing ampicillin (final concentration: 50 μg/ml), and culture was conducted at 37° C. overnight. A single colony of the transformed SOLR was picked up and cultured in LB agar medium containing ampicillin (final concentration: 50 μg/ml) at 37° C., and plasmd DNA having an insert of interest was purified using the QIAGEN plasmid Miniprep Kit (QIAGEN).

The purified plasmid was subjected to analysis of the full-length sequence of the insert via primer walking using the T3 primer of SEQ ID NO: 31 and the T7 primer of SEQ ID NO: 32. The gene sequences shown by SEQ ID NOs: 5, 7, 9, 11, and 13 were obtained via the sequence analysis. The nucleotide sequences of the genes and the amino acid sequences thereof (SEQ ID NOs: 6, 8, 10, 12, and 14) were used to perform homology search with the known genes with the use of a homology search program, BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, all the 5 obtained genes were found to encode CAPRIN-1. Sequence identity among the 5 genes was as follows: the nucleotide sequence identity of 100% and the amino acid sequence identity of 99% in regions to be translated into proteins. Sequence identity of the gene with a gene encoding a human homologue was as follows: the nucleotide sequence identity of 94% and the amino acid sequence identity of 97% in regions to be translated into proteins. The nucleotide sequences of the human homologues are shown by SEQ ID NOs: 1 and 3 and the amino acid sequences thereof are shown by SEQ ID NOs: 2 and 4. Sequence identity of the obtained canine gene with a gene encoding a bovine homologue was as follows: the nucleotide sequence identity of 94% and the amino acid sequence identity of 97% in regions to be translated into proteins. The nucleotide sequence of the bovine homologue is shown by SEQ ID NO: 15 and the amino acid sequence thereof is shown by SEQ ID NO: 16. Sequence identity between the gene encoding the human homologue and the gene encoding the bovine homologue was as follows: the nucleotide sequence identity of 94% and the amino acid sequence identity of 93% to 97% in regions to be translated into proteins. Sequence identity of the obtained canine gene with a gene encoding a horse homologue was as follows: the nucleotide sequence identity of 93% and the amino acid sequence identity of 97% in regions to be translated into proteins. The nucleotide sequence of the horse homologue is shown by SEQ ID NO: 17 and the amino acid sequence thereof is shown by SEQ ID NO: 18. Sequence identity between the gene encoding the human homologue and the gene encoding the horse homologue was as follows: the nucleotide sequence identity of 93% and the amino acid sequence identity of 96% in regions to be translated into proteins. Sequence identity of the obtained canine gene with a gene encoding a mouse homologue was as follows: the nucleotide sequence identity of 87% to 89% and the amino acid sequence identity of 95% to 97% in regions to be translated into proteins. The nucleotide sequences of the mouse homologues are shown by SEQ ID NOs: 19, 21, 23, 25, and 27 and the amino acid sequences thereof are shown by SEQ ID NOs: 20, 22, 24, 26, and 28. Sequence identity between a gene encoding a human homologue and a gene encoding a mouse homologue was as follows: the nucleotide sequence identity of 89% to 91% and the amino acid sequence identity of 95% to 96% in regions to be translated into proteins. Sequence identity of the obtained canine gene with a gene encoding a chicken homologue was as follows: the nucleotide sequence identity of 82% and the amino acid sequence identity of 87% in regions to be translated into proteins. The nucleotide sequence of the chicken homologue is shown by SEQ ID NO: 29 and the amino acid sequence thereof is shown by SEQ ID NO: 30. Sequence identity between a gene encoding a human homologue and a gene encoding a chicken homologue was as follows: the nucleotide sequence identity of 81% to 82% and the amino acid sequence identity of 86% in regions to be translated into proteins.

(4) Gene Expression Analysis in Tissue

Expression of the genes obtained in the above-described manner in canine and human normal tissues and various cell strains was examined via reverse transcription-PCR (RT-PCR). Reverse transcription was carried out in the following manner. Specifically, total RNA was extracted from 50 to 100 mg of tissue samples and 5 to 10×10⁶ cell strains with the use of the TRIzol reagent (Invitrogen) in accordance with the protocol included therein. cDNA was synthesized with the use of the extracted total RNA using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) in accordance with the protocol included therein. PCR was carried out using primers specific for the obtained genes (shown by SEQ ID NOs: 33 and 34) in the following manner. Specifically, reagents (0.25 μl of the sample prepared via reverse transcription, 2 μM each of the primers, 0.2 mM each of dNTPs, and 0.65 U of ExTaq polymerase (Takara Shuzo Co., Ltd.)) were mixed with an accompanying buffer to be adjusted to match the total volune of 25 μl. The resultant was subjected to the reaction of 30 cycles at 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds using the Thermal Cycler (BIO RAD). The above gene-specific primers were used to amplify a region from nucleotides 206 to 632 of the nucleotide sequence shown by SEQ ID NO: 5 (i.e., the canine CAPRIN-1 gene) and a region from nucleotides 698 to 1124 of the nucleotide sequence shown by SEQ ID NO: 1 (i.e., the human CAPRIN-1 gene). For comparison, GAPDH-specific primers (as shown by SEQ ID NOs: 35 and 36) were simultaneously used. As a result, potent expression was observed in a healthy canine testis tissue, and expression was observed in canine breast cancer and adenocarcinoma tissue, shown in FIG. 1. Further, expression of the human homologue of the obtained gene was also examined, and expression thereof was observed only in the testis in the case of normal tissue, as with the case of the canine CAPRIN-1 gene. However, expression thereof was detected in a wide variety of cancer cell lines, such as breast cancer, brain tumor, leukemia, lung cancer, and esophagus cancer cell lines, and, in particular, expression thereof was observed in many breast cancer cell lines. The results demonstrate that CAPRIN-1 expression is not observed in normal tissues other than the testis tissue, however, CAPRIN-1 is expressed in many cancer cells, and breast cancer cell lines, in particular.

In FIG. 1, Reference number 1 on the vertical axis shows the expression pattern of the gene identified above and Reference number 2 shows the expression pattern of the control GAPDH gene.

(5) Immunohistochemical Staining

(5)-1: CAPRIN-1 Expression in Mouse and Canine Normal Tissue

Mice (Balb/c, female) and dogs (beagle dogs, female) were exsanguinated under ether anesthesia and under ketamine/isoflurane anesthesia, the abdominal cavity was opened, and organs (stomach, liver, eyeball, thymus, muscle, bone marrow, uterus, small intestine, esophagus, heart, kidney, salivary gland, large intestine, mammary gland, brain, lung, skin, adrenal gland, ovary, pancreas, spleen, and bladder) were independently transferred to a PBS-containing 10-cm dish. The organs were incised in PBS and immobilized with 0.1 M phosphate buffer containing 4% paraformaldehyde (PFA) (pH 7.4) under reflux conditions overnight. A reflux solution was discarded, the tissue surfaces of the organs were rinsed with PBS, a PBS solution containing 10% sucrose was introduced into a 50-ml centrifuge tube, and tissues were introduced therein and shaken at 4° C. for 2 hours using a rotor. The resultant was transferred into a PBS solution containing 20% sucrose, it was allowed to stand at 4° C. until the tissues settled, and then it was transferred into a PBS solution containing 30% sucrose and allowed to stand at 4° C. until the tissues settled. The tissues were removed and necessary regions were excised using a surgical scalpel. Subsequently, the OCT compound (Tissue Tek) was applied on the tissue surface, and the tissues were then placed on the Cryomold. After the Cryomold was placed on dry ice and rapidly frozen, the tissues were sliced to a thickness of 10 μm to 20 μm using the cryostat (LEICA), and the tissue slices on glass slides were air-dried with a hair drier for 30 minutes to prepare glass slides having tissue slices thereon. Subsequently, the glass slides were introduced into a staining jar filled with PBS-T (physiological saline containing 0.05% Tween 20), PBS-T was replaced with fresh PBS-T every 5 minutes, and this procedure was repeated 3 times. Extra moisture around the slices was wiped off using Kimwipes, the slices were encircled with Dakopen (DAKO), a MOM mouse Ig blocking reagent (VECTASTAIN) was placed on the mouse tissue as a blocking solution, a PBS-T solution containing 10% fetal bovine serum was placed on the canine tissue as a blocking solution, and they were allowed to stand in a moist chamber at room temperature for 1 hour. Subsequently, a solution adjusted to comprise a monoclonal antibody against CAPRIN-1 having the heavy-chain variable region of SEQ ID NO: 78 and the light-chain variable region of SEQ ID NO: 79 at 10 μg/ml with a blocking solution, which reacts with the cancer cell surface prepared in Reference Example 1, was applied thereon, and the resultant was allowed to stand in a moist chamber at 4° C. overnight. After the glass slides were washed 3 times with PBS-T for 10 minutes, the MOM biotin-labeled anti-IgG antibody (VECTASTAIN) diluted 250-fold with a blocking solution was added and was then allowed to stand in a moist chamber at room temperature for 1 hour. After the slides were washed 3 times with PBS-T for 10 minutes, an avidin-biotin ABC reagent (VECTASTAIN) was applied thereon, and the slides were allowed to stand in a moist chamber at room temperature for 5 minutes. After the slides were washed 3 times with PBS-T for 10 minutes, a DAB color-developing solution (10 mg of DAB+10 μl of 30% H₂O₂/0.05 M Tris-HCl, pH 7.6, 50 ml) was applied thereon, and the slides were allowed to stand in a moist chamber at room temperature for 30 minutes. The slides were rinsed with distilled water, a hematoxylin reagent (DAKO) was applied thereon, and the slides were allowed to stand at room temperature for 1 minute, followed by rinsing with distilled water. The glass slides were successively dipped in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 1 minute each and then allowed to stand in xylene overnight. The glass slides were removed, mounted with Glycergel Mounting Medium (DAKO), and then observed. As a result, weak CAPRIN-1 expression was observed within cells of the salivary gland, kidney, colon, and gastric tissues; however, expression thereof was not observed on cell surfaces, and no expression was observed in tissues derived from other organs.

(5)-2: CAPRIN-1 Expression in Canine Breast Cancer Tissue

The 108 frozen breast cancer tissue specimens of dogs, which had been diagnosed to have malignant breast cancer via pathological diagnosis, were used to prepare slides comprising frozen slices thereon and to carry out immunohistochemical staining with the use of a monoclonal antibody against CAPRIN-1 having the heavy-chain variable region of SEQ ID NO: 78 and the light-chain variable region of SEQ ID NO: 79 in the same manner as described above. As a result, CAPRIN-1 expression was observed in 100 of the 108 specimens (92.5%), and CAPRIN-1 expression was found to be particularly potent on the cancer cell surface with a high grade of atypism.

(5)-3: CAPRIN-1 Expression in Human Breast Cancer Tissue

Immunohistochemical staining was carried out on 188 breast cancer tissue specimens on the paraffin-embedded human breast cancer tissue array (BIOMAX). The human breast cancer tissue array was treated at 60° C. for 3 hours, the array was introduced into a staining jar filled with xylene, xylene was replaced with fresh xylene every 5 minutes, and this procedure was repeated 3 times. Subsequently, the similar procedures were repeated with the use of ethanol and PBS-T instead of xylene. The human breast cancer tissue array was introduced into a staining jar filled with 10 mM citrate buffer containing 0.05% Tween 20 (pH 6.0), the array was treated at 125° C. for 5 minutes, and the array was allowed to stand at room temperature for at least 40 minutes. Extra moisture around the slices was wiped off using Kimwipes, the tissue was encircled with Dakopen, and an adequate amount of Peroxidase Block (DAKO) was added dropwise thereto. After the array was allowed to stand at room temperature for 5 minutes, the array was introduced into a staining jar filled with PBS-T, PBS-T was replaced with fresh PBS-T every 5 minutes, and this procedure was repeated 3 times. A PBS-T solution containing 10% FBS was applied thereon as a blocking solution, and it was allowed to stand in a moist chamber at room temperature for 1 hour. Subsequently, a solution adjusted to comprise a monoclonal antibody against CAPRIN-1 having the heavy-chain variable region of SEQ ID NO: 78 and the light-chain variable region of SEQ ID NO: 79 at 10 μg/ml with a PBS-T solution containing 5% FBS, which reacts with the cancer cell surface prepared in Reference Example 1, was applied thereon, and the array was allowed to stand in a moist chamber at 4° C. overnight. After the array was washed 3 times with PBS-T for 10 minutes, an adequate amount of the Peroxidase Labelled Polymer Conjugated (DAKO) was added dropwise thereto, and the array was then allowed to stand in a moist chamber at room temperature for 30 minutes. After the array was washed 3 times with PBS-T for 10 minutes, a DAB color-developing solution (DAKO) was applied thereon, the array was allowed to stand at room temperature for about 10 minutes, and the color-developing solution was discarded. The array was washed 3 times with PBS-T for 10 minutes, rinsed with distilled water, successively dipped in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 1 minute each, and then allowed to stand in xylene overnight. The glass slides were removed, mounted with Glycergel Mounting Medium (DAKO), and then observed. As a result, potent CAPRIN-1 expression was observed in 138 of the entire 188 breast cancer tissue specimens (73%).

(5)-4: CAPRIN-1 Expression in Human Malignant Brain Tumor

Immunohistochemical staining was carried out on 247 malignant brain tumor tissue specimens on the paraffin-embedded human malignant brain tumor tissue array (BIOMAX), with a monoclonal antibody against CAPRIN-1 having the heavy-chain variable region of SEQ ID NO: 78 and the light-chain variable region of SEQ ID NO: 79 in the same manner as in (5)-3 above. As a result, potent CAPRIN-1 expression was observed in 227 of the entire 247 malignant brain tumor tissue specimens (92%).

(5)-5: CAPRIN-1 Expression in Human Breast Cancer-Metastasized Lymph Node

Immunohistochemical staining was carried out on 150 breast cancer-metastasized lymph node tissue specimens on the paraffin-embedded human breast cancer-metastasized lymph node tissue array (BIOMAX), with a monoclonal antibody against CAPRIN-1 having the heavy-chain variable region of SEQ ID NO: 78 and the light-chain variable region of SEQ ID NO: 79 in the same manner as in (5)-3 above. As a result, potent CAPRIN-1 expression was observed in 136 of the entire 150 breast cancer-metastasized lymph node specimens (90%). CAPRIN-1 expression was found to be potent in cancer tissue metastasized from breast cancer.

Reference Example 1 Preparation of Monoclonal Antibody Against CAPRIN-1

100 μg of the antigen protein of SEQ ID NO: 2 prepared in Example 2 (human CAPRIN-1) was mixed with the equivalent amount of the MPL+TDM adjuvant (Sigma Corporation), and the mixture was used as an antigen solution per mouse. The antigen solution was administered intraperitoneally to 6-week-old Balb/c mice (Japan SLC, Inc.), and the solution was administered 3 more times every week. The spleen removed 3 days after the final immunization was sandwiched between two sterilized glass slides, grounded, washed with PBS(−) (Nissui), and centrifuged at 1,500 rpm for 10 minutes, and the supernatant was removed. This procedure was repeated 3 times to obtain spleen cells. The obtained spleen cells were mixed with the SP2/0 mouse myeloma cells (purchased from ATCC) at 10:1, a PEG solution prepared by mixing 200 μl of RPMI 1640 medium containing 10% FBS with 800 μl of PEG1500 (Boehringer) heated at 37° C. was added thereto, and the resultant was allowed to stand for 5 minutes to perform cell fusion. The resultant was centrifuged at 1,700 rpm for 5 minutes, the supernatant was removed, cells were suspended in 150 ml of RPMI 1640 medium containing 15% FBS to which 2% equivalents of a HAT solution (Gibco) had been added (HAT selection medium), and 100 μl each thereof was seeded per well of 15 96-well plates (Nunc). Culture was conducted for 7 days at 37° C. in 5% CO₂ to obtain hybridomas resulting from fusion of spleen cells with myeloma cells.

Hybridomas were selected using the binding affinity of the antibody produced by the resulting hybridomas to the CAPRIN-1 protein as the indicator. The CAPRIN-1 protein solution (1 μg/ml) prepared in Example 2 was added to the 96-well plate in amounts of 100 μl per well, and the plate was allowed to stand at 4° C. for 18 hours. Wells were washed 3 times with PBS-T, a 0.5% bovine serum albumin (BSA) solution (Sigma Corporation) was added in amounts of 400 μl per well, and the plate was allowed to stand at room temperature for 3 hours. The solution was removed, wells were washed 3 times with 400 μl of PBS-T per well, the culture supernatants of the hybridomas obtained above were added in amounts of 100 μl per well, and the plate was allowed to stand at room temperature for 2 hours. After wells were washed 3 times with PBS-T, the HRP-labeled anti-mouse IgG (H+L) antibody (Invitrogen) diluted 5,000-fold with PBS was added in amounts of 100 μl per well, and the plate was allowed to stand at room temperature for 1 hour. After wells were washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added in amounts of 100 μl per well, and the plate was allowed to stand for 15 to 30 minutes to perform a color-developing reaction. After color was developed, 1N sulfuric acid was added in amounts of 100 μl per well to terminate the reaction, and the absorbance at 450 nm and that at 595 nm were assayed using an absorption spectrometer. As a result, a plurality of hybridomas producing antibodies with high absorbance values were selected.

The selected hybridomas were added to a 96-well plate in amounts of 0.5 cells per well and cultured. One week later, some hybridomas were observed to form single colonies in wells. The cells in such wells were further cultured, and hybridomas were selected using the binding affinity of antibodies produced from the cloned hybridomas to the CAPRIN-1 protein as the indicator. The CAPRIN-1 protein solution (1 μg/ml) prepared in Example 2 was added to a 96-well plate in amounts of 100 μl per well and the plate was allowed to stand at 4° C. for 18 hours. After the wells were washed 3 times with PBS-T, a 0.5% BSA solution was added in amounts of 400 μl per well, and the plate was allowed to stand at room temperature for 3 hours. The solution was removed, the wells were washed 3 times with 400 μl of PBS-T per well, the culture supernatants of the hybridomas obtained above were added in amounts of 100 μl per well, and the plate was allowed to stand at room temperature for 2 hours. After the wells were washed 3 times with PBS-T, the HRP-labeled anti-mouse IgG (H+ L) antibody (Invitrogen) diluted 5,000-fold with PBS was added in amounts of 100 μl per well, and the plate was allowed to stand at room temperature for 1 hour. After the wells were washed 3 times with PBS-T, a TMB substrate solution (Thermo) was added in amounts of 100 μl per well, and the plate was allowed to stand for 15 to 30 minutes to perform a color-developing reaction. After color was developed, 1N sulfuric acid was added in amounts of 100 μl per well to terminate the reaction, and the absorbance at 450 nm and that at 595 nm were assayed using an absorption spectrometer. As a result, a plurality of hybridoma strains producing monoclonal antibodies showing reactivity with the CAPRIN-1 protein were obtained, the culture supernatant of hybridomas were purified using a protein G carrier to obtain 150 monoclonal antibodies binding to the CAPRIN-1 protein.

Subsequently, monoclonal antibodies having reactivity with the surfaces of the breast cancer cells expressing CAPRIN-1 were selected from among the above monoclonal antibodies. Specifically, 10⁶ cells of human breast cancer cell line, MDA-MB-231V, were centrifuged in a 1.5-ml microcentrifuge tube, 100 μl of the hybridoma supernatants prepared above was added thereto, and the resultants were allowed to stand on ice for 1 hour. After the plate was washed with PBS, the FITC-labeled goat anti-mouse IgG antibody (Invitrogen) diluted 500-fold with PBS containing 0.1% fetal bovine serum was added, and the plate was allowed to stand on ice for 1 hour. After the plate was washed with PBS, fluorescence intensity was measured using the FACSCalibur (Becton Dickinson). Separately, as a control, the same procedure was carried out except for adding a medium instead of the antibodies. As a result, 11 monoclonal antibodies exhibiting stronger fluorescence intensity than the control; i.e., 11 monoclonal antibodies reacting with the breast cancer cell surfaces, were selected. The sequence of the heavy-chain variable region of one of such monoclonal antibodies is shown by SEQ ID NO: 78 and that of the light-chain variable region thereof is shown by SEQ ID NO: 79.

Example 2 Preparation of Canine and Human Novel Cancer Antigen Proteins

(1) Preparation of Recombinant Protein

A recombinant protein was prepared using the gene of SEQ ID NO: 5 obtained in Example 1 in the manner described below. Reagents (the vector (1 μl) prepared from the phagemid solution obtained in Example 1 and subjected to sequence analysis, 0.4 μM each of two types of primers containing NdeI and KpnI restriction sites (SEQ ID NOs: 37 and 38), 0.2 mM of dNTPs, and 1.25 U of PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.)) were mixed with the accompanying buffer to be adjusted to match the total volume of 50 μl. The resultant was subjected to the reaction of 30 cycles at 98° C. for 10 seconds and 68° C. for 1.5 minutes using the Thermal Cycler (BIO RAD). The two above types of primers were used to amplify a region encoding the full-length amino acid sequence of SEQ ID NO: 6. After PCR was carried out, the amplified DNA was electrophoresed on 1% agarose gel, and a DNA fragment of about 1.4 kbp was purified using the QIAquick Gel Extraction Kit (QIAGEN).

The purified DNA fragment was ligated to the pCR-Blunt cloning vector (Invitrogen). The resultant was transformed into E. coli, the plasmid was recovered, and the amplified gene fragment was confirmed to match the sequence of interest via sequencing. The plasmid, which had been found to match the sequence of interest, was treated with the NdeI and KpnI restriction enzymes, the resultant was purified with the QIAquick Gel Extraction Kit, and the gene sequence of interest was inserted into an E. coli expression vector (pET30b, Novagen) treated with the NdeI and KpnI restriction enzymes. Use of this vector enables production of a His-tag-fused recombinant protein. The plasmid was transformed into E. coli BL21 (DE3) and induced with 1 mM IPTG to express the protein of interest in E. coli.

Separately, a recombinant protein of the canine homologous gene was prepared using the gene of SEQ ID NO: 7 in the manner described below. Reagents (cDNA (1 μl), expression of which was confirmed via RT-PCR among the tissue or cellular cDNA prepared in Example 1, 0.4 μM each of two types of primers containing NdeI and KpnI restriction sites (SEQ ID NOs: 39 and 40), 0.2 mM of dNTPs, and 1.25 U of PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.)) were mixed with an accompanying buffer to be adjusted to match the total volume of 50 μl. The resultant was subjected to the reaction of 30 cycles at 98° C. for 10 seconds and 68° C. for 2.5 minutes, using the Thermal Cycler (BIO RAD). The two above types of primers were used to amplify a region encoding the full-length amino acid sequence of SEQ ID NO: 8. After PCR was carried out, the amplified DNA was electrophoresed on 1% agarose gel, and a DNA fragment of about 2.2 kbp was purified using the QIAquick Gel Extraction Kit (QIAGEN).

The purified DNA fragment was ligated to the pCR-Blunt cloning vector (Invitrogen). The resultant was transformed into E. coli, the plasmid was recovered, and the amplified gene fragment was confirmed to match the sequence of interest via sequencing. The plasmid, which had been found to match the sequence of interest, was treated with the NdeI and KpnI restriction enzymes, the resultant was purified with the QIAquick Gel Extraction Kit, and the gene sequence of interest was inserted into an E. coli expression vector (pET30b, Novagen) treated with the NdeI and KpnI restriction enzymes. Use of this vector enables production of a His-tag-fused recombinant protein. The plasmid was transformed into E. coli BL21 (DE3) and induced with 1 mM IPTG to express the protein of interest in E. coli.

A recombinant protein of the human homologous gene was prepared using the gene of SEQ ID NO: 1 in the manner described below. Reagents (cDNA (1 μl), expression of which was confirmed via RT-PCR among the tissue or cellular cDNA prepared in Example 1, 0.4 μM each of two types of primers containing SacI and XhoI restriction sites (SEQ ID NOs: 41 and 42), 0.2 mM of dNTPs, and 1.25 U of PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.)) were mixed with an accompanying buffer to be adjusted to match the total volume of 50 μl. The resultant was subjected to the reaction of 30 cycles at 98° C. for 10 seconds and 68° C. for 2.5 minutes, using the Thermal Cycler (BIO RAD). The two above types of primers were used to amplify a region encoding the full-length amino acid sequence of SEQ ID NO: 2. After PCR was carried out, the amplified DNA was electrophoresed on 1% agarose gel, and a DNA fragment of about 2.1 kbp was purified using the QIAquick Gel Extraction Kit (QIAGEN).

The purified DNA fragment was ligated to the pCR-Blunt cloning vector (Invitrogen). The resultant was transformed into E. coli, the plasmid was recovered, and the amplified gene fragment was confirmed to match the sequence of interest via sequencing. The plasmid, which had been found to match the sequence of interest, was treated with the SacI and XhoI restriction enzymes, the resultant was purified with the QIAquick Gel Extraction Kit, and the gene sequence of interest was inserted into an E. coli expression vector (pET30a, Novagen) treated with the SacI and XhoI restriction enzymes. Use of this vector enables production of a His-tag-fused recombinant protein. The plasmid was transformed into E. coli BL21 (DE3) and induced with 1 mM IPTG to express the protein of interest in E. coli.

(2) Purification of Recombinant Protein

Recombinant E. coli cells expressing the gene of SEQ ID NO: 1, 5, or 7 obtained above were cultured in LB medium containing 30 μg/ml kanamycin at 37° C. until the absorbance at 600 nm became around 0.7, isopropyl-β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM, and culture was conducted at 37° C. for 4 hours. Thereafter, the cells were centrifuged at 4,800 rpm for 10 minutes and harvested. The cell pellet was suspended in phophate-buffered physiological saline, the resultant was centrifuged at 4,800 rpm for an additional 10 minutes, and the cells were then washed.

The cells were suspended in phophate-buffered physiological saline and ultrasonically disrupted on ice. The solution of ultrasonically disrupted E. coli cells was centrifuged at 6,000 rpm for 20 minutes, the resulting supernatant was designated as a soluble fraction, and the precipitate was designated as an insoluble fraction.

The soluble fraction was added to a nickel-chelating column prepared in accordance with a conventional technique (carrier: Chelateing Sepharose™ Fast Flow (GE Health Care); column volume: 5 ml; equilibration buffer: 50 mM hydrochloride buffer, pH 8.0). An unadsorbed fraction was washed out with 10 column volumes of 50 mM hydrochloride buffer (pH 8.0) and 20 mM phosphate buffer containing 20 mM imidazole (pH 8.0), followed by elution with 6 bed volumes of 20 mM phosphate buffer containing 100 mM imidazole (pH 8.0) immediately thereafter. The fraction eluted with 20 mM phosphate buffer containing 100 mM imidazole (pH 8.0), in which elution of the protein of interest had been confirmed via Coomassie staining, was added to a strong-anion-exchange column (carrier: Q Sepharose™ Fast Flow (GE Health Care); column volume: 5 ml; 20 mM phosphate buffer (pH 8.0) as an equilibration buffer). An unadsorbed fraction was washed out with 10 column volumes of 20 mM phosphate buffer (pH 7.0) and 20 mM phosphate buffer containing 200 mM sodium chloride (pH 7.0), followed by elution with 5 bed volumes of 20 mM phosphate buffer containing 400 mM sodium chloride (pH 7.0) immediately thereafter. The purified fractions of proteins each comprising the amino acid sequences shown by SEQ ID NOs: 2, 6, and 8 were obtained and then designated as materials for administration test.

A fraction (200 μl) of the purified samples obtained by the method described above was dispensed into 1 ml of a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl₂, pH 7.4), 2 μl of enterokinase (Novagen) was added, the resultant was allowed to stand at room temperature overnight, the reaction was allowed to proceed, a His tag was cleaved, and purification was then carried out using the Enterokinase Cleavage Capture Kit (Novagen) in accordance with the accompanying protocols. Subsequently, 1.2 ml of the purified sample obtained by the method described above was subjected to the buffer replacement with phosphate buffered saline (Nissui) via ultrafiltration using Nanosep 10K Omega (Pall), aseptic filtration was carried out through HT Tuffryn Acrodisc (pore size: 0.22 μm, Pall), and the resultant was used for the following experiment.

Example 3 Test of Administration of Recombinant Protein to Cancer-Bearing Dog

(1) Antitumor Evaluation

A cancer-bearing dog (breast cancer) having a tumor on the epidermis was subjected to evaluation of the antitumor effects of the recombinant protein purified above.

The recombinant polypeptide having the amino acid sequence shown by SEQ ID NO: 6 purified in the above-described manner (100 μg, 0.5 ml) was mixed with the equivalent amount of the Freund's incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) to prepare a cancer therapeutic agent. The resulting agent was administered 3 times in total to the regional lymph node in the vicinity of the tumor, as an initial-administration, and 3 days and 7 days thereafter. As a result, a tumor about 86 mm³ in size when the cancer therapeutic agent was administered shrank to 55 mm³, 30 mm³, and 20 mm³ 10 days, 20 days, and 30 days after the initial administration, respectively.

A mixture of 0.5 ml of the recombinant polypeptide having the amino acid sequence shown by SEQ ID NO: 2 and 0.5 ml of the Freund's incomplete adjuvant was administered to another dog having breast cancer 3 times in total in the same manner as described above. Also, 100 μg of canine interleukin 12 was administered together with the agent subcutaneously. As a result, a tumor about 123 mm³ in size when the cancer therapeutic agent was administered completely regressed 45 days after the initial administration of the cancer therapeutic agent.

Further, a mixture of 0.5 ml of the recombinant polypeptide having the amino acid sequence shown by SEQ ID NO: 8 and 0.5 ml of the Freund's incomplete adjuvant was administered to another dog having breast cancer 3 times in total in the same manner as described above. Also, 100 μg of canine interleukin 12 was administered together with the agent subcutaneously. As a result, a tumor about 96 mm³ in size when the cancer therapeutic agent was administered completely regressed 27 days after the initial administration of the cancer therapeutic agent.

(2) Evaluation of Immunity-Inducing Capacity

The blood samples of the clinically affected dogs to which the recombinant polypeptides having the amino acid sequences shown by SEQ ID NO: 6, SEQ ID NO: 2, and SEQ ID NO: 8 had been administered in the administration test conducted in (1) above were obtained before administration and 10 days and 30 days after the initial administration, peripheral blood mononuclear cells were separated in accordance with a conventional technique, and the capacity of the recombinant proteins for immunity induction was evaluated via the ELISpot assay of IFNγ using the peripheral blood mononuclear cells.

70% ethanol was added to a 96-well plate (MultiScreen-IP, MAIPS4510, Millipore) in amounts of 100 μl/well, the plate was allowed to stand for 5 minutes, ethanol was aspirated off, the plate was washed with sterilized water, 200 mM sodium bicarbonate (pH 8.2) was added in amounts of 300 μl/well, the plate was allowed to stand for 5 minutes, sodium bicarbonate was aspirated off, and the plate was washed. Subsequently, the anti-canine interferon γ monoclonal antibody (clone142529, MAB781, R&D) added to 200 mM sodium bicarbonate was added to the plate in amounts of 0.5 μg/well, incubation was carried out at 37° C. overnight, and the primary antibody was solid-phased. After the primary antibody in the solution was aspirated off, a blocking solution (1% BSA-5% sucrose-200 mM sodium bicarbonate, pH 8.2) was added in amounts of 300 μl/well, and incubation was carried out at 4° C. overnight to block the plate. After the blocking solution was aspirated off, RPMI medium containing 10% fetal bovine serum (Invitrogen) was added in amounts of 300 μl/well, the plate was allowed to stand for 5 minutes, and the medium was aspirated off. Thereafter, canine peripheral blood mononuclear cells suspended in RPMI medium containing 10% fetal bovine serum were added to the plate in amounts of 5×10⁵ cells/well, the dog-derived polypeptide or human-derived polypeptide used for administration were added thereto in amounts of 10 μl/well, and culture was conducted at 37° C. in 5% CO₂ for 24 hours to produce interferon γ from immunocytes among peripheral blood mononuclear cells. After culture was conducted, the medium was removed, and wells were washed 6 times with a wash (0.1% Tween 20-200 mM sodium bicarbonate, pH 8.2). The rabbit anti-dog polyclonal antibodies diluted 1,000-fold with the blocking solution were added to the plate in amounts of 100 μl/well and then incubated at 4° C. overnight. After the wells were washed 3 times with the wash, the HRP-labeled anti-rabbit antibodies diluted 1,000-fold with the blocking solution were added to the plate in amounts of 100 μl/well, and the reaction was allowed to proceed at 37° C. for 2 hours. After the wells were washed 3 times with the wash, a color was developed with the aid of a Konica immunostain (Konica), and the wells was washed with water to terminate the reaction. After the reaction was terminated, the membrane was dried, and the number of the developed spots was counted using the KS Elispot (Carl Zeiss). As a result, no spots were detected in the peripheral blood mononuclear cells of clinically affected dogs before polypeptide administration. In the case after polypeptides were administered, however, 13 and 82 spots were detected in the peripheral blood mononuclear cells obtained from the clinically affected dogs to which the recombinant polypeptide having the amino acid sequence shown by SEQ ID NO: 6 had been administered, 10 and 30 days after the administration. In the case of clinically affected dogs to which the recombinant polypeptide having the amino acid sequence shown by SEQ ID NO: 2 had been administered, 53 and 189 spots were detected in the peripheral blood mononuclear cells 10 and 30 days after the administration. In the case of clinically affected dogs to which the recombinant polypeptide having the amino acid sequence shown by SEQ ID NO: 8 had been administered, 32 and 117 spots were detected in the peripheral blood mononuclear cells 10 and 30 days after the administration.

The above results demonstrate that immunocytes that produce interferon γ specifically in response to the recombinant proteins that have been administered are induced in clinically affected dogs to which the recombinant polypeptide had been administered. The results also demonstrate that the immune reactions in which such immunocytes play a central role, produce the antitumor effects described in (1) above.

Example 4 Antitumor Effects of DNA Vaccines

A recombinant plasmid was prepared using the gene of SEQ ID NO: 19 in the following manner. Reagents (cDNA (1 μl) extracted in the same manner as in Example 1(4) from the mouse colorectal cancer cell line (CT26, purchased from ATCC) in which CAPRIN-1 expression had been observed, 0.4 μM each of two types of primers (shown by SEQ ID NOs: 80 and 81), 0.2 mM of dNTPs, and 1.25 U of PrimeSTAR HS polymerase) were mixed with an accompanying buffer to be adjusted to match the total volume of 50 μl. The resultant was subjected to PCR of 30 cycles at 98° C. for 10 seconds, 55° C. for 15 seconds, and 72° C. for 4 minutes, using a Thermal Cycler. The two above types of primers were used to amplify a region encoding the full-length amino acid sequence of SEQ ID NO: 20. After PCR was carried out, the amplified DNA was electrophoresed on 1% agarose gel, and a DNA fragment of about 2,100 bp was purified using the QIAquick Gel Extraction Kit.

The purified DNA fragment was ligated to the pCR-Blunt cloning vector (Invitrogen). The resultant was transformed into E. coli, the plasmid was recovered, the sequence of the fragment was analyzed, and a plasmid having an amplified gene fragment matching with the sequence of interest was obtained. The plasmid was treated with the EcoRI restriction enzyme, the resultant was purified with the QIAquick Gel Extraction Kit, and the gene sequence of interest was inserted into a mammalian expression vector (pcDNA3.1, Invitrogen) treated with the EcoRI restriction enzyme in accordance with a conventional technique.

50 μg of gold particles (Bio Rad), 100 μl of spermidine (SIGMA), and 100 μl of 1M CaCl₂ were added to 100 μg of plasmid DNA prepared above, the mixture was agitated by a vortex, and the resultant was allowed to stand at room temperature for 10 minutes (hereafter referred to as “gold-DNA particles”). After the mixture was centrifuged at 3,000 rpm for 1 minute, the supernatant was discarded, and the particles were washed 3 times with 100% ethanol. 100% ethanol (6 ml) was added to the gold-DNA particles, the resultant was thoroughly agitated by a vortex, and the gold-DNA particles were introduced into the Tefzel Tubing (Bio Rad) and precipitated on the wall. Ethanol in the Tefzel Tubing to which the gold-DNA particles had adrered was air-dried, and the dried tube was cut in a length suitable for gene gun applications.

CT26 cells were transplanted subcutaneously into the dorsal regions of 20 Balb/c mice (Japan SLC, Inc.) in amounts of 10⁶ cells/mouse and grown to a tumor diameter of approximately 7 mm. Thereafter, the tube prepared above was fixed on the gene gun, the cells were percutaneously administered into the abdominal cavities of shaved mice at a pressure of 400 psi using a pure helium gas (the amount of plasmid DNA inoculated is 2 μg/mouse), and the antitumor effects were evaluated.

As a result, tumors became enlarged in 10 control mice to which empty plasmids comprising no CAPRIN-1 genes had been administered, and all the 10 mice died 63 days after tumor transplantation. In the case of 10 mice to which plasmids comprising the CAPRIN-1 genes inserted therein had been administered, however, tumors completely regressed by 25 days after tumor transplantation, and all mice remained alive 63 days after tumor transplantation when all control mice died.

Example 5 Induction of Peptide Epitope-Reactive CD8+ T Cells

(1) Prediction of HLA-A0201-Binding Peptide Motif and HLA-A24-Binding Peptide Motif

The amino acid sequence information of the human CAPRIN-1 polypeptide was obtained from the GenBank. In order to predict an HLA-A0201-binding peptide motif and an HLA-A24-binding peptide motif, the amino acid sequence of the human CAPRIN-1 polypeptide was analyzed via a computer prediction program using known BIMAS softwares (available at http://bimas.dcrtnih.gov/molbio/hla_bind/), 29 types of peptides shown by SEQ ID NO: 43 to SEQ ID NO: 71 predicted to be capable of binding to the HLA-A0201 molecules and 5 types of peptides shown by SEQ ID NO: 72 to SEQ ID NO: 76 predicted to be capable of binding to the HLA-A24 molecules were selected.

(2) Induction of Peptide Epitope-Reactive CD8+ T Cells

The peripheral blood was separated from an HLA-A0201-positive healthy individual, overlaid on the lymphocyte separation medium (OrganonpTeknika, Durham, N.C.), and centrifuged at 1,500 rpm at room temperature for 20 minutes. A PBMC-containing fraction was recovered and washed 3 (or more) times in cold phosphate buffer to obtain peripheral blood mononuclear cells (PBMCs). The obtained PBMCs were suspended in 20 ml of AIM-V medium (Life Technologies) and adhered to a culture flask (Falcon) at 37° C. in 5% CO₂ for 2 hours. The nonadhered cells were used for T cell preparation, and the adhered cells were used for dendritic cell preparation.

The adhered cells were cultured in AIM-V medium in the presence of IL-4 (1,000 U/ml) and GM-CSF (1,000 U/ml). The medium was exchanged with another AIM-V medium to which IL-4 (1,000 U/ml), GM-CSF (1,000 U/ml), IL-6 (1,000 U/ml, Genzyme, Cambridge, Mass.), IL-1β (10 ng/ml, Genzyme, Cambridge, Mass.), and TNF-α (10 ng/ml, Genzyme, Cambridge, Mass.) had been added 6 days later, culture was conducted for an additional 2 days, and the obtained nonadhered cell population was used as dendritic cells.

The prepared dendritic cells were suspended in AIM-V medium at a cell density of 1×10⁶ cells/ml, the peptides shown by SEQ ID NO: 43 to SEQ ID NO: 71 predicted to be capable of binding to the HLA-A0201 molecules selected in (1) above were added thereto at 10 μg/ml, and culture was conducted using a 96-well plate at 37° C. in 5% CO₂ for 4 hours. After culture was conducted, the cells were irradiated with X-rays (3,000 rad), washed with AIM-V medium, suspended in AIM-V medium containing 10% human AB serum (Nabi, Miami, Fla.), IL-6 (1,000 U/ml), and IL-12 (10 ng/ml, Genzyme, Cambridge, Mass.), and added to a 24-well plate in amounts of 1×10⁵ cells/well. Further, the prepared T cell population was added in amounts of 1×10⁶ cells/well and cultured at 37° C. in 5% CO₂. The culture supernatants were discarded 7 days later, the dendritic cells treated with peptides obtained in the above-described manner and then irradiated with X-rays were suspended in AIM-V medium containing 10% human AB serum (Nabi, Miami, Fla.), IL-7 (10 U/ml, Genzyme, Cambridge, Mass.), and IL-2 (10 U/ml, Genzyme, Cambridge, Mass.) (cell density: 1×10⁵ cells/ml), the cells were added to a 24-well plate in amounts of 1×10⁵ cells/well, and culture was further conducted. After such procedure was repeated 4 to 6 times every 7 days, the stimulated T cells were recovered, and CD8+ T cell induction was confirmed via flow cytometry.

Regarding the peptides of SEQ ID NO: 72 to SEQ ID NO: 76 predicted to be capable of binding to the HLA-A24 molecules, induction of peptide epitope-reactive CD8+ T cells was also attempted in the same manner as described above with the use of dendritic cells and T cell population induced from the peripheral blood of an HLA-A24-positive healthy individual.

As a negative control, a peptide having a sequence outside the scope of the present invention (SEQ ID NO: 77) was used.

Example 6 Determination of Cytotoxic T Cell Antigen Epitope

(1) Capacity for IFN-γ Production

In order to determine the specificity of the T cells that had been observed to grow to peptide epitopes, among the T cells induced in Example 5(2), 5×10³ T cells were added to 5×10⁴ HLA-A0201 molecules-expressing T2 cells (Salter R D et al., Immunogenetics, 21: 235-246, 1985, T2 cells were purchased from ATCC) pulsed with the peptides predicted to be capable of binding to the HLA-A0201 molecules (the peptides were added to AIM-V medium at 10 μg/ml and cultured at 37° C. in 5% CO₂ for 4 hours), and the resultant was cultured on a 96-well plate in AIM-V medium containing 10% human AB serum for 24 hours. The supernatants were collected after culturing, and the amount of IFN-γ produced was measured via ELISA. As a result, IFN-γ production was observed more in the culture supernatants obtained from the wells in which T2 cells pulsed with the peptides of SEQ ID NO: 43 to SEQ ID NO: 71 were used than in the culture supernatants obtained from the wells in which T2 cells not pulsed with peptides were used (FIG. 2). The results demonstrate that the above peptides are T cell epitope peptides capable of specifically stimulating the proliferation of HLA-A0201+ CD8+ T cells to induce IFN-γ production.

In the same manner as described above, specificity of the peptide epitope-reactive CD8+ T cells induced with the use of the peptides of SEQ ID NO: 72 to SEQ ID NO: 76 in Example 5(2) to the peptide epitopes was determined in the following manner. Specifically, IFN-γ production level by the T cells against the peptide-pulsed JTK-LCL cells expressing the HLA-A24 molecules (the JTK-LCL cells were purchased from RIKEN, Japan) was measured via ELISA. As a result, IFN-γ production was observed more in the culture supernatants obtained from the wells in which JTK-LCL cells pulsed with the peptides of SEQ ID NO: 72 to SEQ ID NO: 76 were used than in the culture supernatants obtained from the wells in which JTK-LCL cells not pulsed with peptides were used (FIG. 3). The results demonstrate that the peptides of SEQ ID NO: 72 to SEQ ID NO: 76 are T cell epitope peptides capable of specifically stimulating the proliferation of HLA-A24+ CD8+ T cells to induce IFN-γ production.

(2) Cytotoxicity Evaluation

Subsequently, whether or not peptides of SEQ ID NO: 43 to SEQ ID NO: 71 used in the present invention were presented on the HLA-A0201 molecules on the HLA-A0201+ tumor cells expressing the human CAPRIN-1 polypeptide and whether or not the CD8+ T cells stimulated with the peptides were capable of destroying the HLA-A0201+ tumor cells expressing the human CAPRIN-1 polypeptide were examined. 10⁶ cells of U-87MG human glioma cells (purchased from ATCC), which were verified to express the human CAPRIN-1 polypeptide, were collected in a 50-ml centrifuge tube, 100 μCi chromium-51 was added, and incubation was carried out at 37° C. for 2 hours. Thereafter, the cells were washed 3 times with RPMI medium (Gibco) containing 10% fetal bovine serum (hereafter referred to as “FBS,” Gibco) and added to a 96-well V-bottom plate in amounts of 10³ cells/well. Further, 5×10⁴ HLA-A0201+ and peptide epitope-reactive CD8+ T cells stimulated with the peptides suspended in RPMI medium containing 10% FBS were added, and culture was conducted at 37° C. in 5% CO₂ for 4 hours. After the culture, the amount of chromium-51 in the culture supernatant released from the disrupted tumor cells was measured to determine the cytotoxic activity of the peptide-stimulated CD8+ T cells. As a result, the peptide-stimulated HLA-A0201+ CD8+ T cells were found to have cytotoxic activity against U-87MG cells (FIG. 4). In contrast, the CD8+ T cells induced with the use of a negative control peptide (SEQ ID NO: 77) did not show cytotoxic activity. Thus, the peptides used in the present invention (i.e., peptides of SEQ ID NO: 43 to SEQ ID NO: 71) were found to be presented on the HLA-A0201 molecules on the HLA-A0201+ tumor cells expressing the human CAPRIN-1 polypeptide. Further, the peptides were also found to be capable of inducing CD8+ cytotoxic T cells that could disrupt such tumor cells.

Subsequently, whether or not peptides of SEQ ID NO: 72 to SEQ ID NO: 76 were presented on the HLA-A24 molecules on the HLA-A24+ tumor cells expressing the human CAPRIN-1 polypeptide and whether or not the CD8+ T cells stimulated with the peptides were capable of disrupting the HLA-A24+ tumor cells expressing the human CAPRIN-1 polypeptide were examined in the same manner as described above. Chromium-51 was incorporated into the HLA-A24+ JTK-LCL cells expressing the human CAPRIN-1 polypeptide, HLA-A24+ and peptide epitope-reactive CD8+ T cells were added, culture was conducted, and the amount of chromium-51 in the culture supernatant released from the disrupted cells was measured. As a result, the HLA-A24+ CD8+ T cells stimulated with the peptides of SEQ ID NO: 72 to SEQ ID NO: 76 were found to have the cytotoxic activity against the JTK-LCL cells (FIG. 5). Thus, the peptides of SEQ ID NO: 72 to SEQ ID NO: 76 were found to be presented on the HLA-A24 molecules on the HLA-A24+ cells expressing the human CAPRIN-1 polypeptide, and the peptides were found to be capable of inducing CD8+ cytotoxic T cells capable of disrupting such cells. The CD8+ T cells induced with the use of a negative control peptide (SEQ ID NO: 77) did not exhibit cytotoxicity.

In order to determine the cytotoxic activity, 5×10⁴ CD8+ T cells stimulated and induced by the peptides used in the present invention were mixed with 10³ U-87MG or JTK-LCL cells into which chromium-51 had been incorporated, cultured for 4 hours, and the amount of chromium-51 released into the medium after culture was measured. The cytotoxic activity used herein means the cytotoxic activity of the CD8+ T cells against the U-87MG cells or the JTK-LCL cells (i.e., target cells) determined according to the following equation*. Cytotoxic activity (%)=amount of chromium-51 released from U-87MG or JTK-LCL cell upon addition of CD8+ T cell/amount of chromium-51 released from target cell upon addition of 1N hydrochloric acid×100  Equation*

INDUSTRIAL APPLICABILITY

The present invention is industrially useful for the purpose of treatment and prevention of cancer.

This description includes part or all of the disclosure of the description and/or drawings of Japanese Patent Application No. 2008-202065, to which the present application claims the priority. Also, all publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 31: T3 primer

SEQ ID NO: 32: T7 primer

SEQ ID NOs: 33 to 34: primers

SEQ ID NOs: 35 to 36: GAPDH primers

SEQ ID NOs: 37 to 42 and 80 to 81: primers 

The invention claimed is:
 1. A method for inducing regression of a cancer selected from the group consisting of breast cancer, brain tumor, leukemia, lymphoma, lung cancer or esophagus cancer by inducing immunity against the cancer, said method comprising: administering to a human or dog individual having the cancer which expresses CAPRIN-1 polypeptide on the cell surface at least one polypeptide having T-cell immunity-inducing activity and selected from SEQ ID NO: 2 or 4, or a recombinant vector comprising a polynucleotide encoding said polypeptide and capable of expressing said polypeptide in vivo to induce T cell immunity against the cancer and thereby cause regression of the cancer.
 2. The method according to claim 1, wherein one or plural types of said polypeptides are administered.
 3. The method according to claim 1, wherein the polypeptide is an agent for treating an antigen-presenting cell.
 4. The method according to claim 1, wherein the animal is a human.
 5. The method according to claim 1, wherein said method further comprises administering an immunopotentiating agent.
 6. The method according to claim 5, wherein the immunopotentiating agent is at least one adjuvant or cytokine selected from the group consisting of Freund's incomplete adjuvant, Montanide, poly IC and a derivative thereof, CpG oligonucleotide, interleukin 12, interleukin 18, interferon α, interferon β, interferon ω, interferon γ, and Flt3 ligand. 